Emeritus Faculty, Acad Council, Biochemistry
I closed my laboratory when I retired in 1998. I continue to do research, chiefly in collaboration with Franc Avbelj, on problems of protein folding energetics, especially peptide backbone solvation, and to write reviews.
Kauzmann's explanation of how the hydrophobic factor drives protein folding is reexamined. His explanation said that hydrocarbon hydration shells are formed, possibly of clathrate water, and they explain why hydrocarbons have uniquely low solubilities in water. His explanation was not universally accepted because of skepticism about the clathrate hydration shell. A revised version is given here in which a dynamic hydration shell is formed by van der Waals (vdw) attraction, as proposed in 1985 by Jorgensen et al. [Jorgensen WL, Gao J, Ravimohan C (1985) J Phys Chem 89:3470-3473]. The vdw hydration shell is implicit in theories of hydrophobicity that contain the vdw interaction between hydrocarbon C and water O atoms. To test the vdw shell model against the known hydration energetics of alkanes, the energetics should be based on the Ben-Naim standard state (solute transfer between fixed positions in the gas and liquid phases). Then the energetics are proportional to n, the number of water molecules correlated with an alkane by vdw attraction, given by the simulations of Jorgensen et al. The energetics show that the decrease in entropy upon hydration is the root cause of hydrophobicity; it probably results from extensive ordering of water molecules in the vdw shell. The puzzle of how hydrophobic free energy can be proportional to nonpolar surface area when the free energy is unfavorable and the only known interaction (the vdw attraction) is favorable, is resolved by finding that the unfavorable free energy is produced by the vdw shell.
View details for DOI 10.1073/pnas.1414556111
View details for Web of Science ID 000341625600030
View details for PubMedID 25157156
In the new view, hydrophobic free energy is measured by the work of solute transfer of hydrocarbon gases from vapor to aqueous solution. Reasons are given for believing that older values, measured by solute transfer from a reference solvent to water, are not quantitatively correct. The hydrophobic free energy from gas-liquid transfer is the sum of two opposing quantities, the cavity work (unfavorable) and the solute-solvent interaction energy (favorable). Values of the interaction energy have been found by simulation for linear alkanes and are used here to find the cavity work, which scales linearly with molar volume, not accessible surface area. The hydrophobic free energy is the dominant factor driving folding as judged by the heat capacity change for transfer, which agrees with values for solvating hydrocarbon gases. There is an apparent conflict with earlier values of hydrophobic free energy from studies of large-to-small mutations and an explanation is given.
View details for DOI 10.1016/j.febslet.2013.01.006
View details for Web of Science ID 000317188700010
View details for PubMedID 23337880
The exquisite side chain close-packing in the protein core and at binding interfaces has prompted a conviction that packing selectivity is the primary mechanism for molecular recognition in folding and/or binding reactions. Contrary to this view, molten globule proteins can adopt native topology and bind targets tightly and specifically in the absence of side chain close-packing. The molten globule is a highly dynamic form with native-like secondary structure and a loose protein core that admits solvent. The related (but still controversial) dry molten globule is an expanded form of the native protein with largely intact topology but a tighter protein core that excludes solvent. Neither form retains side chain close-packing, and therefore both structure and function must result from other factors, assuming that the reality of the dry molten globule is accepted. This simplifying realization calls for a re-evaluation of established models.
View details for DOI 10.1016/j.sbi.2012.11.004
View details for Web of Science ID 000315832700002
View details for PubMedID 23237704
The hydrophobic free energy in current use is based on transfer of alkane solutes from liquid alkanes to water, and it has been argued recently that these values are incorrect and should be based instead on gas-liquid transfer data. Hydrophobic free energy is measured here by gas-liquid transfer of hydrocarbon gases from vapor to water. The new definition reduces more than twofold the values of the apparent hydrophobic free energy. Nevertheless, the newly defined hydrophobic free energy is still the dominant factor that drives protein folding as judged by ΔCp, the change in heat capacity, found from the free energy change for heat-induced protein unfolding. The ΔCp for protein unfolding agrees with ΔCp values for solvating hydrocarbon gases and disagrees with ΔCp for breaking peptide hydrogen bonds, which has the opposite sign. The ΔCp values for the enthalpy of liquid-liquid and gas-liquid transfer are similar. The plot of free energy against the apparent solvent-exposed surface area is given for linear alkanes, but only for a single conformation, the extended conformation, of these flexible-chain molecules. The ability of the gas-liquid hydrophobic factor to predict protein stability is tested and reasonable agreement is found, using published data for the dependences on temperature of the unfolding enthalpy of ribonuclease T1 and the solvation enthalpies of the nonpolar and polar groups.
View details for DOI 10.1073/pnas.1220825110
View details for Web of Science ID 000314558100026
View details for PubMedID 23319615
Hydrophobic free energy for protein folding is currently measured by liquid-liquid transfer, based on an analogy between the folding process and the transfer of a nonpolar solute from water into a reference solvent. The second part of the analogy (transfer into a nonaqueous solvent) is dubious and has been justified by arguing that transfer out of water probably contributes the major part of the free energy change. This assumption is wrong: transfer out of water contributes no more than half the total, often less. Liquid-liquid transfer of the solute from water to liquid alkane is written here as the sum of 2 gas-liquid transfers: (i) out of water into vapor, and (ii) from vapor into liquid alkane. Both gas-liquid transfers have known free energy values for several alkane solutes. The comparable values of the two different transfer reactions are explained by the values, determined in 1991 for three alkane solutes, of the cavity work and the solute-solvent interaction energy. The transfer free energy is the difference between the positive cavity work and the negative solute-solvent interaction energy. The interaction energy has similar values in water and liquid alkane that are intermediate in magnitude between the cavity work in water and in liquid alkane. These properties explain why the transfer free energy has comparable values (with opposite signs) in the two transfers. The current hydrophobic free energy is puzzling and poorly defined and needs a new definition and method of measurement.
View details for DOI 10.1073/pnas.1203720109
View details for Web of Science ID 000304090600043
View details for PubMedID 22529345
Hydrogen exchange (HX) is recognized today as one of the most powerful and versatile tools available to protein scientists, especially for studying protein conformational change. This short history traces the beginnings of the HX method and the basic problems that faced the founders. Protein HX began as a simple idea with a straightforward goal, but the first experiments revealed both the unexpected complexity of the subject and the potential power of the method for probing deep into how proteins work. By 1972, the chemistry of the exchange reaction in peptides began to be well understood, but the challenge of getting and interpreting data on HX for individual peptide NH protons in proteins remained for decades longer.
View details for DOI 10.1002/prot.23039
View details for Web of Science ID 000292924500001
View details for PubMedID 21557321
The amide III region of the peptide infrared and Raman spectra has been used to determine the relative populations of the three major backbone conformations (P(II), β, and α(R)) in 19 amino acid dipeptides. The results provide a benchmark for force field or other methods of predicting backbone conformations in flexible peptides. There are three resolvable backbone bands in the amide III region. The major population is either P(II) or β for all dipeptides except Gly, whereas the α(R) population is measurable but always minor (≤ 10%) for 18 dipeptides. (The Gly ϕ,ψ map is complex and so is the interpretation of the amide III bands of Gly.) There are substantial differences in the relative β and P(II) populations among the 19 dipeptides. The band frequencies have been assigned as P(II), 1,317-1,306 cm(-1); α(R), 1,304-1,294 cm(-1); and β, 1,294-1,270 cm(-1). The three bands were measured by both attenuated total reflection spectroscopy and by Raman spectroscopy. Consistent results, both for band frequency and relative population, were obtained by both spectroscopic methods. The β and P(II) bands were assigned from the dependence of the (3)J(H(N),H(α)) coupling constant (known for all 19 dipeptides) on the relative β population. The P(II) band assignment agrees with one made earlier from Raman optical activity data. The temperature dependences of the relative β and P(II) populations fit the standard model with Boltzmann-weighted energies for alanine and leucine between 30 and 60 °C.
View details for DOI 10.1073/pnas.1017317108
View details for Web of Science ID 000286804700012
View details for PubMedID 21205907
A novel analysis of the enthalpy of protein unfolding is proposed and used to test for a desolvation penalty when hydrogen-bonded peptide groups are desolvated via folding. The unfolding enthalpy has three components, (1) the change when peptide hydrogen bonds are broken and the exposed -CO and -NH groups are solvated, (2) the change when protein-protein van der Waals interactions are broken and replaced by protein-water van der Waals interactions, and (3) the change produced by the hydrophobic interaction when nonpolar groups in the protein interior (represented as a liquid hydrocarbon) are transferred to water. A key feature of the analysis is that the enthalpy change from the hydrophobic interaction goes through 0 at 22 °C according to the liquid hydrocarbon model. Protein unfolding enthalpies are smaller at 22 °C than the enthalpy change for unfolding an alanine peptide helix. Data in the literature indicate that the van der Waals contribution to the unfolding enthalpy is considerably larger than the unfolding enthalpy itself at 22 °C, and therefore, a sizable desolvation penalty is predicted. Such a desolvation penalty was predicted earlier from electrostatic calculations of a stabilizing interaction between water and the hydrogen-bonded peptide group.
View details for DOI 10.1021/jp107111f
View details for Web of Science ID 000284990700023
View details for PubMedID 20961078
Understanding the secondary structure of peptides is important in protein folding, enzyme function, and peptide-based drug design. Previous studies of synthetic Ala-based peptides (>12 a.a.) have demonstrated the role for charged side chain interactions involving Glu/Lys or Glu/Arg spaced three (i, i + 3) or four (i, i + 4) residues apart. The secondary structure of short peptides (<9 a.a.), however, has not been investigated. In this study, the effect of repetitive Glu/Lys or Glu/Arg side chain interactions, giving rise to E-R/K helices, on the helicity of short peptides was examined using circular dichroism. Short E-R/K-based peptides show significant helix content. Peptides containing one or more E-R interactions display greater helicity than those with similar E-K interactions. Significant helicity is achieved in Arg-based E-R/K peptides eight, six, and five amino acids long. In these short peptides, each additional i + 3 and i + 4 salt bridge has substantial contribution to fractional helix content. The E-R/K peptides exhibit a strongly linear melt curve indicative of noncooperative folding. The significant helicity of these short peptides with predictable dependence on number, position, and type of side chain interactions makes them an important consideration in peptide design.
View details for DOI 10.1002/pro.469
View details for Web of Science ID 000282716900019
View details for PubMedID 20669185
New experimental results show that either gain or loss of close packing can be observed as a discrete step in protein folding or unfolding reactions. This finding poses a significant challenge to the conventional two-state model of protein folding. Results of interest involve dry molten globule (DMG) intermediates, an expanded form of the protein that lacks appreciable solvent. When an unfolding protein expands to the DMG state, side chains unlock and gain conformational entropy, while liquid-like van der Waals interactions persist. Four unrelated proteins are now known to form DMGs as the first step of unfolding, suggesting that such an intermediate may well be commonplace in both folding and unfolding. Data from the literature show that peptide amide protons are protected in the DMG, indicating that backbone structure is intact despite loss of side-chain close packing. Other complementary evidence shows that secondary structure formation provides a major source of compaction during folding. In our model, the major free-energy barrier separating unfolded from native states usually occurs during the transition between the unfolded state and the DMG. The absence of close packing at this barrier provides an explanation for why phi-values, derived from a Brønsted-Leffler plot, depend primarily on structure at the mutational site and not on specific side-chain interactions. The conventional two-state folding model breaks down when there are DMG intermediates, a realization that has major implications for future experimental work on the mechanism of protein folding.
View details for DOI 10.1002/prot.22803
View details for Web of Science ID 000281541800001
View details for PubMedID 20635344
Recent calorimetric measurements of the solvation enthalpies of some dipeptide analogs confirm our earlier prediction that the principle of group additivity is not valid for the interaction of the peptide group with water. We examine the consequences for understanding the properties of peptide solvation. A major consequence is that the current value of the peptide-solvation enthalpy, which is a basic parameter in analyzing the energetics of protein folding, is seriously wrong. Electrostatic calculations of solvation-free energies provide an estimate of the size and nature of the error. Peptide hydrogen exchange rates provide an experimental approach for testing the accuracy of the solvation-free energies of peptide groups found by electrostatic calculations. These calculations emphasize that ignoring electrostatic interactions with neighboring NHCO groups should be a major source of error. Results in 1972 for peptide hydrogen exchange rates demonstrate that peptide-solvation-free energies are strongly affected by adjoining NHCO groups. In the past, the effect of adjoining peptide groups on the exchange rate of a peptide NH proton was treated as an inductive effect. The effect can be calculated, however, by an electrostatic model with fixed partial charges and a continuum solvent.
View details for DOI 10.1073/pnas.0813018106
View details for Web of Science ID 000263844100031
View details for PubMedID 19202077
My research began with theory and methods for ultracentrifugal studies of proteins, first at the University of Wisconsin, Madison, with Bob Alberty and Jack Williams, then at Oxford University with A.G. ("Sandy") Ogston, and finally back at Wisconsin with Williams and Lou Gosting. In 1959 I joined Arthur Kornberg's Biochemistry Department at Stanford University. Our first work was physical studies of DNA replication and then DNA physical chemistry, and DNA studies ended with the energetics of DNA twisting. In 1971 we began to search for protein folding intermediates by fast-reaction methods. We found the slow-folding and fast-folding forms of unfolded ribonuclease A, which led to the understanding that proline isomerization is sometimes part of the folding process. Using hydrogen exchange as a probe, we found the rapid formation of secondary structure during folding and used this to provide an NMR pulse labeling method for determining structures of folding intermediates. Our studies of peptide helices provided basic helix-coil parameters, also evidence for hierarchic folding, and further indicated that peptide hydrogen bonds are important in the energetics of folding.
View details for DOI 10.1146/annurev.biophys.37.032807.125948
View details for Web of Science ID 000258107500002
View details for PubMedID 18573070
The energetics of protein folding determine the 3D structure of a folded protein. Knowledge of the energetics is needed to predict the 3D structure from the amino acid sequence or to modify the structure by protein engineering. Recent developments are discussed: major factors are reviewed and auxiliary factors are discussed briefly. Major factors include the hydrophobic factor (burial of non-polar surface area) and van der Waals interactions together with peptide hydrogen bonds and peptide solvation. The long-standing model for the hydrophobic factor (free energy change proportional to buried non-polar surface area) is contrasted with the packing-desolvation model and the approximate nature of the proportionality between free energy and apolar surface area is discussed. Recent energetic studies of forming peptide hydrogen bonds (gas phase) are reviewed together with studies of peptide solvation in solution. Closer agreement is achieved between the 1995 values for protein unfolding enthalpies in vacuum given by Lazaridis-Archontis-Karplus and Makhatadze-Privalov when the solvation enthalpy of the peptide group is taken from electrostatic calculations. Auxiliary factors in folding energetics include salt bridges and side-chain hydrogen bonds, disulfide bridges, and propensities to form alpha-helices and beta-structure. Backbone conformational entropy is a major energetic factor which is discussed only briefly for lack of knowledge.
View details for DOI 10.1016/j.jmb.2007.05.078
View details for Web of Science ID 000248509100001
View details for PubMedID 17582437
The principle of group additivity is a standard feature of analyses of the energetics of protein folding, but it is known that it may not always be valid for the polar peptide group. The neighboring residue effect shows that group additivity is not strictly valid for a heteropeptide. We show here that group additivity fails seriously for peptide groups close to either peptide end, even for a homopeptide that has blocked end groups with no formal charges involved. The failure of group additivity is caused by the electrostatic character of the solvation of peptide polar groups and is illustrated with values of the electrostatic solvation free energy (ESF) calculated by DelPhi. Solvation free energies and enthalpies are known experimentally for monoamides and are often used to model the solvation of peptide groups, but ESF results show that monoamide values are very different from those of peptide groups. A main cause of the difference is that peptide solvation depends on the dipole-dipole interactions made between adjacent peptide groups, which vary with peptide conformation. Ligands that interact with the peptide backbone by an electrostatic mechanism could show a similar peptide end effect, and hydrogen exchange results from the literature confirm that exchange rates are position-dependent close to peptide ends.
View details for DOI 10.1002/prot.20756
View details for Web of Science ID 000236419700005
View details for PubMedID 16288449
The preferences of amino acid residues for ,psi backbone angles vary strikingly among the amino acids, as shown by the backbone angle found from the (3)J(H(alpha),H(N)) coupling constant for short peptides in water. New data for the (3)J(H(alpha),H(N)) values of blocked amino acids (dipeptides) are given here. Dipeptides exhibit the full range of coupling constants shown by longer peptides such as GGXGG and dipeptides present the simplest system for analyzing backbone preferences. The dipeptide coupling constants are surprisingly close to values computed from the coil library (conformations of residues not in helices and not in sheets). Published coupling constants for GGXGG peptides agree closely with dipeptide values for all nonpolar residues and for some polar residues but not for X = D, N, T, and Y, which are probably affected by polar side chain-backbone interactions in GGXGG peptides. Thus, intrinsic backbone preferences are already determined at the dipeptide level and remain almost unchanged in GGXGG peptides and are strikingly similar in the coil library of conformations from protein structures. The simplest explanation for the backbone preferences is that backbone conformations are strongly affected by electrostatic dipole-dipole interactions in the peptide backbone and by screening of these interactions with water, which depends on nearby side chains. Strong backbone electrostatic interactions occur in dipeptides. This is shown by calculations both of backbone electrostatic energy for different conformers of the alanine dipeptide in the gas phase and by electrostatic solvation free energies of amino acid dipeptides.
View details for DOI 10.1073/pnas.0510420103
View details for Web of Science ID 000235094300023
View details for PubMedID 16423894
The NMR chemical shifts of certain atomic nuclei in proteins ((1)H(alpha),(13)C(alpha), and (13)C(beta)) depend sensitively on whether or not the amino acid residue is part of a secondary structure (alpha-helix, beta-sheet), and if so, whether it is helix or sheet. The physical origin of the different chemical shifts of atomic nuclei in alpha-helices versus beta-sheets is a problem of long standing. We report that the chemical shift contributions arising from secondary structure (secondary structure shifts) depend strongly on the extent of exposure to solvent. This behavior is observed for (1)H(alpha), (13)C(alpha), and (13)C(beta) (sheet), but not for(13)C(beta) (helix), whose secondary structure shifts are small. When random coil values are subtracted from the chemical shifts of all(1)H(alpha) nuclei (Pro residues excluded) and the residual chemical shifts are summed to plot the mean values against solvent exposure, the results give a funnel-shaped curve that approaches a small value at full-solvent exposure. When chemical shifts are plotted instead against E(local), the electrostatic contribution to conformational energy produced by local dipole-dipole interactions, a well characterized dependence of (1)H(alpha) chemical shifts on E(local) is found. The slope of this plot varies with both the type of amino acid and the extent of solvent exposure. These results indicate that secondary structure shifts are produced chiefly by the electric field of the protein, which is screened by water dipoles at residues in contact with solvent.
View details for DOI 10.1073/pnas.0407969101
View details for Web of Science ID 000225803400017
View details for PubMedID 15574491
Unfolded peptides in water have some residual structure that may be important in the folding process, and the nature of the residual structure is currently of much interest. There is a neighboring residue effect on backbone conformation, discovered in 1997 from measurements of (3)J(HN alpha) coupling constants. The neighboring residue effect appears also in the "coil library" of Protein Data Bank structures of residues not in alpha-helix and not in beta-structure. When a neighboring residue (i - 1 or i + 1) belongs to class L (aromatic and beta-branched amino acids, FHITVWY) rather than class S (all others, G and P excluded), then the backbone angle of residue i is more negative for essentially all amino acids. Calculated values of peptide solvation (electrostatic solvation free energy, ESF) predict basic properties of the neighboring residue effect. We show that L amino acids reduce the solvation of neighboring peptide groups more than S amino acids. When tripeptides from the coil library are excised to allow solvation, the central residues have more negative values of
View details for DOI 10.1073/pnas.0404050101
View details for Web of Science ID 000223000200024
View details for PubMedID 15254296
The "coil library," consisting of the phi, psi values of residues outside secondary structure in high-resolution protein structures, has chiefly the beta, alpha(R), alpha(L), and polyproline II backbone conformations. In denatured proteins, the 20 aa have different average values of the (3)J(HN)alpha coupling constant, related to the backbone angle phi by the Karplus relation. Average (3)J(HN)alpha values obtained from the distributions of phi, g(phi), of the coil library agree with NMR results, and so the coil library can be and is being used to model denatured proteins. Here, Monte Carlo simulations of backbone conformations in denatured proteins are used to test two physics-based models: the random coil model of Brant and Flory [(1965) J. Am. Chem. Soc. 87, 2788-2791 and 2791-2800] and an electrostatic screening model (ESM) that includes electrostatic solvation. The random coil model represents hindered rotation about phi and psi backbone angles, nonbonded interactions, and dipole-dipole interactions. In the ESM, the nonbonded interactions term is replaced by the use of hard sphere repulsion and allowed regions in the Ramachadran maps. These models were tested by using the amino acid sequences of three small proteins. There are two main conclusions: (i) The g(phi) distributions of the coil library contain detailed, specific information, so that prediction of the g(phi) distributions of the different amino acids is a demanding test of the energy function. (ii) The ESM is partly successful in predicting the g(phi) distributions. Electrostatic solvation is primarily responsible, and steric clash between pairs of atoms connected by torsion angles is not responsible.
View details for DOI 10.1073/pnas.1031522100
View details for Web of Science ID 000182939400034
View details for PubMedID 12709596
The H-bond inventory approach is used commonly to interpret data involving changes in the number or types of protein hydrogen bonds. I point out here that this approach gives an incorrect answer either for the standard free energy or enthalpy of the reaction between simple amides and water. On the other hand, an electrostatic solvation approach fits almost within error the polar solvation free energies of small molecules, including amides. The electrostatic solvation approach is used here to discuss the relation between peptide backbone solvation and the enthalpy change for forming an alanine helix.
View details for PubMedID 12488001
View details for PubMedID 12487983
Very short alanine peptide helices can be studied in a fixed-nucleus, helix-forming system [Siedlicka, M., Goch, G., Ejchart, A., Sticht, H. & Bierzynski, A. (1999) Proc. Natl. Acad. Sci. USA 96, 903-908]. In a 12-residue sequence taken from an EF-hand protein, the four C-terminal peptide units become helical when the peptide binds La(3+), and somewhat longer helices may be made by adding alanine residues at the C terminus. The helices studied here contain 4, 8, or 11 peptide units. Surprisingly, these short fixed-nucleus helices remain almost fully helical from 4 to 65 degrees C, according to circular dichroism results reported here, and in agreement with titration calorimetry results reported recently. These peptides are used here to define the circular dichroism properties of short helices, which are needed for accurate measurement of helix propensities. Two striking properties are: (i) the temperature coefficient of mean peptide ellipticity depends strongly on helix length; and (ii) the intensity of the signal decreases much less rapidly with helix length, for very short helices, than supposed in the past. The circular dichroism spectra of the short helices are compared with new theoretical calculations, based on the experimentally determined direction of the NV(1) transition moment.
View details for DOI 10.1073/pnas.232591399
View details for Web of Science ID 000179530000032
View details for PubMedID 12427967
A sequence of seven alanine residues-too short to form an alpha-helix and whose side chains do not interact with each other-is a particularly simple model for testing the common description of denatured proteins as structureless random coils. The (3)J(HN alpha) coupling constants of individual alanine residues have been measured from 2 to 56 degrees C by using isotopically labeled samples. The results display a thermal transition between different backbone conformations, which is confirmed by CD spectra. The NMR results suggest that polyproline II is the dominant conformation at 2 degrees C and the content of beta strand is increased by approximately 10% at 55 degrees C relative to that at 2 degrees C. The polyproline II conformation is consistent with recent studies of short alanine peptides, including structure prediction by ab initio quantum mechanics and solution structures for both a blocked alanine dipeptide and an alanine tripeptide. CD and other optical spectroscopies have found structure in longer "random coil" peptides and have implicated polyproline II, which is a major backbone conformation in residues within loop regions of protein structures. Our result suggests that the backbone conformational entropy in alanine peptides is considerably smaller than estimated by the random coil model. New thermodynamic data confirm this suggestion: the entropy loss on alanine helix formation is only 2.2 entropy units per residue.
View details for DOI 10.1073/pnas.112193999
View details for Web of Science ID 000176775400023
View details for PubMedID 12091708
Data are reported for T(m), the temperature midpoint of the thermal unfolding curve, of ribonuclease A, versus pH (range 2-9) and salt concentration (range 0-1 M) for two salts, Na(2)SO(4) and NaCl. The results show stabilization by sulfate via anion-specific binding in the concentration range 0-0.1 M and via the Hofmeister effect in the concentration range 0.1-1.0 M. The increase in T(m) caused by anion binding at 0.1 M sulfate is 20 degrees at pH 2 but only 1 degree at pH 9, where the net proton charge on the protein is near 0. The 10 degrees increase in T(m) between 0.1 and 1.0 M Na(2)SO(4), caused by the Hofmeister effect, is independent of pH. A striking property of the NaCl results is the absence of any significant stabilization by 0.1 M NaCl, which indicates that any Debye screening is small. pH-dependent stabilization is produced by 1 M NaCl: the increase in T(m) between 0 and 1.0 M is 14 degrees at pH 2 but only 1 degree at pH 9. The 14 degree increase at pH 2 may result from anion binding or from both binding and Debye screening. Taken together, the results for Na(2)SO(4) and NaCl show that native ribonuclease A is stabilized at low pH in the same manner as molten globule forms of cytochrome c and apomyoglobin, which are stabilized at low pH by low concentrations of sulfate but only by high concentrations of chloride.
View details for DOI 10.1110/ps.0205902
View details for Web of Science ID 000176324800020
View details for PubMedID 12070329
Pairs of leucine side chains, spaced either (i,i+3) or (i,i+4), are known to stabilize alanine-based peptide helices, Experiments with new peptide sequences confirm that the (i,i+4) pair interaction is markedly stronger than the (i,i+3) pair interaction. This result is not expected from reported Monte Carlo simulations, which predict that the (i,i+3) interaction is slightly stronger. The interaction strength can be predicted from recently reported measurements of buried non-polar surface area, obtained from structures in the Protein Data Bank: the agreement is reasonable for the (i,i+3) LL interaction but underestimates the (i,i+4) LL interaction. Solvation of peptide groups in the helix backbone may contribute to the different strengths of the two LL pair interactions because different chi(1) leucine rotamers are used and the (i,i+3) pair shields two peptide groups whereas the (i,i+4) pair shields only one. A rough estimate of the backbone solvation effect, based on the difference between the helix propensities of leucine and alanine, agrees with the size of the difference between the (i,i+3) and (i,i+4) leucine pair interactions.
View details for Web of Science ID 000176060000003
View details for PubMedID 12034432
The early events in protein folding are often difficult to track. A hydrophobic collapse, during which nonpolar residues are buried away from the polar solvent, has been proposed, but little is known about the role of this collapse in protein folding. In his Perspective, Baldwin discusses the report by Klein-Seetharaman et al., who shed some light on this issue. A network of hydrophobic clusters stabilizes at least one nonnative interaction in unfolded hen lysozyme. In conjunction with earlier studies, the results suggest that this network has a beneficial effect on the folding of the protein.
View details for PubMedID 11872825
There is a paradox concerning the beta propensities of the amino acids: the amino acids with the highest beta propensities such as valine and isoleucine have the highest tendency to desolvate the peptide backbone, which should result in a loss of stability. Nevertheless, backbone solvation, calculated as electrostatic solvation free energy (ESF), is highly correlated with mutant stability in the zinc-finger system studied by Kim and Berg [Kim, C. A. & Berg, J. M. (1993) Nature (London) 362, 267-270], and valine and isoleucine are among the most stabilizing amino acids. This inverse correlation between stability and ESF can be explained, because the mutant ESF differences in the unfolded protein are larger than in the native protein. Consequently, mutations such as Ala to Val destabilize the unfolded form more than the native protein. By comparing mutant Delta ESF values in isolated beta-strands versus beta-sheets, we conclude that amino acids with high beta propensities should exert their stabilizing effects at early stages in folding. This deduction agrees with the studies by Clarke and coworkers [Lorch, M., Mason, J. M., Clarke, A. R. & Parker, M. J. (1999) Biochemistry 38, 1377-1385, and Lorch, M., Mason, J. M., Sessions, R. B. & Clarke, A. R. (2000) Biochemistry 39, 3480-3485] of the thermodynamics of folding of the beta-sheet protein CD2.d1.
View details for Web of Science ID 000173752500040
View details for PubMedID 11805303
The goal of this study is to use the model system described earlier to make direct measurements of the enthalpy of helix formation at different temperatures. For this we studied model alanine peptides in which helix formation can be triggered by metal (La(3+)) binding. The heat of La(3+) interaction with the peptides at different temperatures is measured by isothermal titration calorimetry. Circular dichroism spectroscopy is used to follow helix formation. Peptides of increasing length (12-, 16-, and 19-aa residues) that contain a La(3+)-binding loop followed by helices of increasing length, are used to separate the heat of metal binding from the enthalpy of helix formation. We demonstrate that (i) the enthalpy of helix formation is -0.9 +/- 0.1 kcal/mol; (ii) the enthalpy of helix formation is independent of the peptide length; (iii) the enthalpy of helix formation does not depend significantly on temperature in the range from 5 to 45 degrees C, suggesting that the heat capacity change on helix formation is very small. Thus, the use of metal binding to induce helix formation has an enormous potential for measuring various thermodynamic properties of alpha-helices.
View details for Web of Science ID 000173752500038
View details for PubMedID 11818561
In native apomyoglobin, His-24 cannot be protonated, although at pH 4 the native protein forms a molten globule folding intermediate in which the histidine residues are readily protonated. The inability to protonate His-24 in the native protein dramatically affects the unfolding/refolding kinetics, as demonstrated by simulations for a simple model. Kinetic data for wild type and for a mutant lacking His-24 are analyzed. The pK(a) values of histidine residues in native apomyoglobin are known from earlier studies, and the average histidine pK(a) in the molten globule is determined from the pH dependence of the equilibrium between the native and molten globule forms. Analysis of the pH-dependent unfolding/refolding kinetics reveals that the average pK(a) of the histidine residues, including His-24, is closely similar in the folding transition state to the value found in the molten globule intermediate. Consequently, protonation of His-24 is not a barrier to refolding of the molten globule to the native protein. Instead, the normal pK(a) of His-24 in the transition state, coupled with its inaccessibility in the native state, promotes fast unfolding at low pH. The analysis of the wild-type results is confirmed and extended by using the wild-type parameters to fit the unfolding kinetics of a mutant lacking His-24.
View details for Web of Science ID 000168883700036
View details for PubMedID 11353859
The apomyoglobin molten globule has a complex, partly folded structure with a folded A[B]GH subdomain; the factors determining its stability are not yet known in detail. Ala-->Gly mutations, made at solvent-exposed positions, are used to probe the role of helix propensity of individual helices in stabilizing the molten globule. Molten globule stability is measured by reversible urea unfolding, monitored both by circular dichroism and by tryptophan fluorescence. Two-state unfolding is tested by superposition of these two unfolding curves, and stability data are reported only for variants which satisfy the superposition test. Results for sites Q8 in the A helix and E109 in the G helix confirm that the helix propensities of the A and G helices both strongly affect molten globule stability, in contrast to results for the G65A/G73A double mutant which show that changing the helix propensity of the E-helix sequence has no significant stabilizing effect. Changing the helix propensity of the B-helix sequence with the G23A/G25A double mutant affects molten globule stability to an intermediate extent, confirming an earlier report that this mutant has increased stability. These results are consistent with the bipartite structure for the molten globule in which the A, G, and H helices are stably folded, while the long E helix is unfolded and the B helix has intermediate stability. Some differences are found in the shapes of the unfolding curves of different mutants even though they satisfy the superposition test for two-state unfolding, and possible explanations are discussed.
View details for DOI 10.1021/bi010122j
View details for Web of Science ID 000168435100021
View details for PubMedID 11318652
The alanine helix provides a model system for studying the energetics of interaction between water and the helical peptide group, a possible major factor in the energetics of protein folding. Helix formation is enthalpy-driven (-1.0 kcal/mol per residue). Experimental transfer data (vapor phase to aqueous) for amides give the enthalpy of interaction with water of the amide group as approximately -11.5 kcal/mol. The enthalpy of the helical peptide hydrogen bond, computed for the gas phase by quantum mechanics, is -4.9 kcal/mol. These numbers give an enthalpy deficit for helix formation of -7.6 kcal/mol. To study this problem, we calculate the electrostatic solvation free energy (ESF) of the peptide groups in the helical and beta-strand conformations, by using the delphi program and parse parameter set. Experimental data show that the ESF values of amides are almost entirely enthalpic. Two key results are: in the beta-strand conformation, the ESF value of an interior alanine peptide group is -7.9 kcal/mol, substantially less than that of N-methylacetamide (-12.2 kcal/mol), and the helical peptide group is solvated with an ESF of -2.5 kcal/mol. These results reduce the enthalpy deficit to -1.5 kcal/mol, and desolvation of peptide groups through partial burial in the random coil may account for the remainder. Mutant peptides in the helical conformation show ESF differences among nonpolar amino acids that are comparable to observed helix propensity differences, but the ESF differences in the random coil conformation still must be subtracted.
View details for Web of Science ID 000089566100024
View details for PubMedID 10984522
The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 degrees C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 degrees C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.
View details for Web of Science ID 000088376100009
View details for PubMedID 10933499
Our aim is to determine whether the disulfide bonds of alpha-lactalbumin account for the lack of cooperative folding behavior reported for some molten globule variants, in contrast to the highly cooperative folding reported for the pH 4 molten globule of apomyoglobin. Two different alpha-lactalbumin genetic constructs are studied: [28-111], which has a single disulfide bond connecting two segments of the alpha-helix domain, and [all-Ala], which has no disulfide bonds. The superposition test used earlier to probe for cooperative folding of the apomyoglobin molten globule is used to determine whether there is an important difference in folding cooperativity between the molten globules of [28-111] and [all-Ala]. The [all-Ala] construct behaves in the same manner as the apomyoglobin molten globule: its folding satisfies the superposition test in the three sets of anion conditions studied, and anions stabilize it against urea unfolding. The [28-111] construct behaves differently in both respects: the folding of its molten globule does not satisfy the superposition test in two of the three sets of anion conditions, and anions barely affect its stability. The 28-111 disulfide bond stabilizes the molten globule substantially, as expected from earlier work. Comparison of the unfolding transition curves monitored by circular dichroism also demonstrates that [28-111] folds in a less cooperative manner than [all-Ala]: the unfolding curve of [28-111] is significantly broader. Moreover, the unfolding curves indicate that [28-111] has a lower helix content than [all-Ala]. Consequently, the 28-111 bond constrains the folding behavior of the molten globule and weakens its cooperativity of folding.
View details for Web of Science ID 000082868500067
View details for PubMedID 10500168
Submillisecond mixing experiments and tryptophan fluorescence spectroscopy are used to address two questions raised in earlier stopped-flow studies of the folding and unfolding kinetics of sperm whale apomyoglobin. A study of the pH 4 folding intermediate (I) revealed, surprisingly, that its folding and unfolding kinetics are measurable and fit the two-state model except for a possible burst phase in unfolding. Submillisecond mixing experiments confirm the unfolding burst phase and show that its properties are consistent with the recently discovered interconversion between two forms of I, Ia equilibrium Ib. In urea-induced unfolding, Ib is converted to Ia before Ia unfolds, and the unfolding kinetics of Ia fit the two-state model when the burst phase is assigned to Ib-->Ia. The second question is whether the Ia, Ib intermediates accumulate transiently when the native protein (N) unfolds to the acid unfolded form (U). Earlier work showed that Ia and Ib accumulate when U refolds to N at pH 6.0 and the results fit the linear folding pathway U equilibrium Ia equilibrium Ib equilibrium N. We report here that either or both Ia and Ib accumulate transiently when N unfolds to U at pH 2.7 and that the position of the rate-limiting step in the pathway changes between unfolding at pH 2. 7 and refolding at pH 6.0. In unfolding as in refolding, we do not detect a fast track that bypasses the Ia, Ib intermediates.
View details for Web of Science ID 000082789700021
View details for PubMedID 10497035
An earlier theoretical study predicted that specific ion pair interactions between neighboring helices should be important in stabilizing myoglobin. To measure these interactions in sperm whale myoglobin, single mutations were made to disrupt them. To obtain reliable DeltaG values, conditions were found in which the urea induced unfolding of holomyoglobin is reversible and two-state. The cyanomet form of myoglobin satisfies this condition at pH 5, 25 degrees C. The unfolding curves monitored by far-UV CD and Soret absorbance are superimposable and reversible. None of the putative ion pairs studied here makes a large contribution to the stability of native myoglobin. The protein stability does decrease somewhat between 0 and 0.1 M NaCl, however, indicating that electrostatic interactions contribute favorably to myoglobin stability at pH 5.0. A previous mutational study indicated that the net positive charge of the A[B]GH subdomain of myoglobin is an important factor affecting the stability of the pH 4 folding intermediate and potential ion pairs within the subdomain do not contribute significantly to its stability. One of the assumptions made in that study is tested here: replacement of either positively or negatively charged residues outside the A[B]GH subdomain has no significant effect on the stability of the pH 4 molten globule.
View details for Web of Science ID 000081768900028
View details for PubMedID 10423259
We report an enthalpic factor involved in determining helix propensities of nonpolar amino acids. Thermal unfolding curves of the five 13-residue peptides, Ac-KA4XA4KGY-NH2 (X = Ala, Leu, Ile, Val, Gly), have been measured by using CD in water/trifluoroethanol (TFE) mixtures. The peptide helix contents show that the rank order of helix propensities changes with temperature: although Ala has the highest helix propensity at 0 degrees C in all TFE concentrations, it is lower than Leu, Ile, and Val at 50 degrees C in 20% TFE. This change is attributed to shielding by nonpolar side chains of the interaction between water and polar groups in the helix backbone for the following reasons. (i) Helix content is directly related to helix propensity for these designed peptides because side-chain-side-chain interactions are absent. (ii) The change in rank order with temperature is enthalpic in origin: in water, the apparent enthalpy of helix formation calculated from the thermal unfolding curves varies widely among the five peptides and has the same rank order as the helix propensities at 0 degrees C. The rank order does not result from burial of nonpolar surface area because the calculated heat capacity change (DeltaCp) on helix formation is opposite in sign from the expected DeltaCp. (iii) A nonpolar side chain can exclude water from interacting with helix polar groups, according to calculations of water-accessible surface area, and the polar interaction between water and peptide polar groups is entirely enthalpic, as shown by amide transfer data.
View details for Web of Science ID 000080130200038
View details for PubMedID 10220396
Alanine-based peptides of defined sequence and length show measurable helix contents, allowing them to be used as a model system both for analyzing the mechanism of helix formation and for investigating the contributions of side-chain interactions to protein stability. Extensive characterization of many peptide sequences with varying amino acid contents indicates that the favorable helicity of alanine-based peptides can be attributed to the large helix-stabilizing propensity of alanine. Based on their analysis of alanine-rich sequences N-terminally linked to a synthetic helix-inducing template, Kemp and coworkers [Kemp, D. S., Boyd, J. G. & Muendel, C. C. (1991) Nature (London) 352, 451-454; Kemp, D. S., Oslick, S. L. & Allen, T. J. (1996) J. Am. Chem. Soc. 118, 4249-4255] argue that alanine is helix-indifferent, however, and that the favorable helix contents of alanine-based peptides must have some other explanation. Here, we show that the helix contents of template-nucleated sequences are influenced strongly by properties of the template-helix junction. A model in which the helix propensities of residues at the template-peptide junction are treated separately brings the results from alanine-based peptides and template-nucleated helices into agreement. The resulting model provides a physically plausible resolution of the discrepancies between the two systems and allows the helix contents of both template-nucleated and standard peptide helices to be predicted by using a single set of helix propensities. Helix formation in both standard peptides and template-peptide conjugates can be attributed to the large intrinsic helix-forming tendency of alanine.
View details for Web of Science ID 000079507900062
View details for PubMedID 10097097
On exposure to mildly acidic conditions, apomyoglobin forms a partially folded intermediate, I. The A, B, G, and H helices are significantly structured in this equilibrium intermediate, whereas the remainder of the protein is largely unfolded. We report here the effects of mutations at helix pairing sites on the stability of I in three classes of mutants that: (i) truncate hydrophobic side chains in native helix packing sites, (ii) truncate hydrophobic side chains not involved in interhelical contacts, and (iii) extend hydrophobic side chains at residues not involved in interhelical contacts. Class I mutants significantly decrease the stability and cooperativity of folding of the intermediate. Class II and III mutants show smaller effects on stability and have little effect on cooperativity. Qualitatively similar results to those found in I were obtained for all three classes of mutants in native myoglobin (N), demonstrating that hydrophobic burial is fairly specific to native helix packing sites in I as well as in N. These results suggest that hydrophobic burial along native-like interhelical contacts is important for the formation of the cooperatively folded intermediate.
View details for Web of Science ID 000078956600038
View details for PubMedID 10051585
We measured the folding and unfolding kinetics of mutants for a simple protein folding reaction to characterize the structure of the transition state. Fluorescently labeled S-peptide analogues combine with S-protein to form ribonuclease S analogues: initially, S-peptide is disordered whereas S-protein is folded. The fluorescent probe provides a convenient spectroscopic probe for the reaction. The association rate constant, kon, and the dissociation rate constant, koff, were both determined for two sets of mutants. The dissociation rate constant is measured by adding an excess of unlabeled S-peptide analogue to a labeled complex (RNaseS*). This strategy allows kon and koff to be measured under identical conditions so that microscopic reversibility applies and the transition state is the same for unfolding and refolding. The first set of mutants tests the role of the alpha-helix in the transition state. Solvent-exposed residues Ala-6 and Gln-11 in the alpha-helix of native RNaseS were replaced by the helix destabilizing residues glycine or proline. A plot of log kon vs. log Kd for this series of mutants is linear over a very wide range, with a slope of -0.3, indicating that almost all of the molecules fold via a transition state involving the helix. A second set of mutants tests the role of side chains in the transition state. Three side chains were investigated: Phe-8, His-12, and Met-13, which are known to be important for binding S-peptide to S-protein and which also contribute strongly to the stability of RNaseS*. Only the side chain of Phe-8 contributes significantly, however, to the stability of the transition state. The results provide a remarkably clear description of a folding transition state.
View details for Web of Science ID 000078956600040
View details for PubMedID 10051587
The folding reactions of some small proteins show clear evidence of a hierarchic process, whereas others, lacking detectable intermediates, do not. Evidence from folding intermediates and transition states suggests that folding begins locally, and that the formation of native secondary structure precedes the formation of tertiary interactions, not the reverse. Some notable examples in the literature have been interpreted to the contrary. For these examples, we have simulated the local structures that form when folding begins by using the LINUS program with nonlocal interactions turned off. Our results support a hierarchic model of protein folding.
View details for Web of Science ID 000079406500009
View details for PubMedID 10098403
The folding reactions of some small proteins show clear evidence of a hierarchic process, whereas others, lacking detectable intermediates, do not. Nevertheless, we argue that both classes fold hierarchically and that folding begins locally. If this is the case, then the secondary structure of a protein is determined largely by local sequence information. Experimental data and theoretical considerations support this argument. Part I of this article reviews the relationship between secondary structures in proteins and their counterparts in peptides.
View details for Web of Science ID 000079406300008
View details for PubMedID 10087919
A modified pulse-chase experiment is applied to determine if the native-like intermediate IN of ribonuclease A is on or off-pathway. The 1H label retained in the native protein is compared when separate samples of 1H-labeled IN and unfolded protein are allowed to fold to native in identical conditions. The solvent is 2H2O and the pH* is such that the unfolded protein rapidly exchanges its peptide NH protons with solvent, and IN does not. If IN is on-pathway, more 1H-label will be retained in the test sample starting with IN than in the control sample starting with unfolded protein. The results show that IN is a productive (on-pathway) intermediate. Application of the modified pulse-chase experiment to the study of rapidly formed folding intermediates may be possible when a rapid mixing device is used.
View details for Web of Science ID 000076637500013
View details for PubMedID 9784375
The impact of folding funnels and folding simulations on the way experimentalists interpret results is examined. The image of the transition state has changed from a unique species that has a strained configuration, with a correspondingly high free energy, to a more ordinary folding intermediate, whose balance between limited conformational entropy and stabilizing contacts places it at the top of the free energy barrier. Evidence for a broad transition barrier comes from studies showing that mutations can change the position of the barrier. The main controversial issue now is whether populated folding intermediates are productive on-pathway intermediates or dead-end traps. Direct experimental evidence is needed. Theories suggesting that populated intermediates are trapped in a glasslike state are usually based on mechanisms which imply that trapping would only be extremely short-lived (e.g., nanoseconds) in water at 25 degrees C. There seems to be little experimental evidence for long-lived trapping in monomers, if folding aggregates are excluded. On the other hand, there is good evidence for kinetic trapping in dimers. alpha-Helix formation is currently the fastest known process in protein folding, and incipient helices are present at the start of folding. Fast helix formation has the effect of narrowing drastically the choice of folding routes. Thus helix formation can direct folding. It changes the folding metaphor from pouring liquid down a folding funnel to a train leaving a switchyard with only a few choices of exit tracks.
View details for Web of Science ID 000075246200041
View details for PubMedID 9649403
2,2,2-Trifluoroethanol (TFE) is known to stabilize peptide helices by strengthening hydrogen bonds. On the other hand, TFE destabilizes native proteins, as we confirm here, presumably by weakening the hydrophobic interaction. The stability of the pH 4 folding intermediate of apomyoglobin is known to depend both on the strength of the individual A, G, and H helices and on hydrophobic interactions between helices. We ask which effect of TFE dominates in this case: strengthening helices or weakening hydrophobic interactions between helices? Protein stability is measured by denaturant-induced unfolding curves, and two-state unfolding is tested by monitoring both far-UV CD and tryptophan fluorescence emission. Low concentrations of TFE strongly stabilize the pH 4 folding intermediate. Moreover, low concentrations of TFE compensate for helix-destabilizing mutations in the A and G helices. Consequently, enhancing helix propensity, rather than weakening the hydrophobic interaction, is the dominant effect of TFE on the folding intermediate. This result agrees with earlier mutational evidence that helix propensities are very important in determining the stability of the pH 4 intermediate. Although TFE destabilizes native holomyoglobin, as well as native lysozyme and ribonuclease A, nevertheless, TFE stabilizes native apomyoglobin.
View details for Web of Science ID 000074065600005
View details for PubMedID 9636699
The acid-induced unfolding of the pH 4 intermediate of apomyoglobin (I) is described by either of two models: (1) a Monod-Wyman-Changeux-based model (MWC) where salt bridges perturb the pKa values of specific ionizable side chains, causing unfolding of I as these salt bridges are broken at low pH, and (2) the Linderstrom-Lang smeared charge model (L-L), which attributes acid unfolding of I to charge repulsion caused by the accumulation of positive charge on the surface of the protein. Both models fit earlier acid unfolding data well, but they make differing predictions about the effects of electrostatic mutants, which have been made and tested. Deletions of positive charge within I are found to stabilize I, but disruptions of potential salt bridges have little effect. These results show that the acid unfolding of I (I<-->U) is largely caused by generalized charge effects rather than by the loss of specific salt bridges. Acid unfolding of the native form, which is caused largely by a single histidine with a severely depressed pKa, is a sensitive indicator of changes in stability produced by mutations. In contrast, the I <--> U transition is caused by a number of groups with smaller pKa perturbations and both models predict that the pH midpoint of the I right harpoon over left harpoon U transition is an insensitive indicator of stability. This result reconciles previous conflicting results, in urea and acid unfolding studies of hydrophobic contact mutants, by showing that changes in the stability of I are poorly detected by acid unfolding.
View details for Web of Science ID 000074006700024
View details for PubMedID 9601047
Heteronuclear NMR methods are used to study the protonation of histidine and aspartate residues in the acid-induced unfolding of recombinant sperm whale apomyoglobin. The results are combined with fluorescence and circular dichroism measurements of acid-induced unfolding of wild-type and double mutant (H24V/H119F) proteins. They are consistent with a simple model in which the failure to protonate a single buried histidine, H24, is largely responsible for the partial unfolding of native (N) wild-type apomyoglobin to the pH 4 folding intermediate (I). H24 is known to form an unusual interaction in which its side chain is buried and hydrogen-bonded to the side chain of H119. Two-dimensional 1H-15N heteronuclear NMR spectra indicate that H24 is present in the rare delta tautomeric form and remains neutral until N unfolds to I, while H119 becomes protonated before the N --> I reaction occurs. In the H24V/H119F double mutant, all histidines are protonated in N and the N --> I reaction occurs at lower pH. Therefore, the protonation of aspartate and/or glutamate residues must provide an additional driving force for the N to I reaction. Two-dimensional 1H-13C NMR experiments are used to measure the protonation of aspartates in selectively 13C-labeled apomyoglobin; the results indicate that none of the aspartate residues has a strongly depressed pKa in N, as would be expected if it forms a stabilizing salt bridge.
View details for Web of Science ID 000072916600028
View details for PubMedID 9521748
The effects of mutations, temperature, and solvent viscosity on the bimolecular association rate constant (kon) and dissociation rate constant (koff) of the complex (RNaseS*) formed by S-peptide analogues and folded S-protein are reported. An important advantage of this system is that both kon and koff may be measured under identical strongly native conditions, and Kd for the complex may be calculated from the ratio koff/kon (preceding article). The side chains of S-peptide residues His-12 and Met-13 contribute a large fraction of the total interface with S-protein. Changing these residues, either singly or in a double mutant, destabilizes RNaseS* by up to 6 orders of magnitude, but causes no more than a 3-fold decrease in kon. Therefore, nativelike side-chain interactions between these residues and S-protein are not present in the transition state for folding. The absence of side-chain interactions in the transition state is surprising, since it has buried 55% of the total surface area that is buried upon forming RNaseS*, as estimated from the denaturant dependences of kon and koff (preceding article). The temperature dependence of the refolding rate suggests that the transition state for complex formation is stabilized by hydrophobic interactions: 66% of the change in heat capacity on forming RNaseS* occurs in the association reaction, consistent with the estimate of surface area burial from the denaturant studies. The solvent viscosity is varied to determine if the folding reaction is diffusion limited. Because kon, koff, and Kd all can be measured under the same native conditions, the viscosity effect on reaction rates can be separated from the effect of sucrose on the stability of RNaseS*. Both kon and koff are found to be inversely proportional to the solvent viscosity, indicating that the association and dissociation kinetics are diffusion controlled. The stabilizing effect of sucrose on RNaseS* appears as a reduction in koff.
View details for Web of Science ID 000072299900053
View details for PubMedID 9485405
The bimolecular association rate constant (kon) and dissociation rate constant (koff) of the complex between fluorescein-labeled S-peptide analogues and folded S-protein are reported. This is the first kinetic study of a protein folding reaction in which most of the starting material is already folded and only a small part (one additional helix) becomes ordered; it provides a folding landscape with a small conformational entropy barrier, and one in which kinetic traps are unlikely. Refolding and unfolding are measured under identical strongly native conditions, and the reaction is found to be two-state at low reactant concentrations. The dissociation constant (Kd) of the complex and the properties of the transition state may be calculated from the rate constants without extrapolation. The folded complex is formed fast (kon = 1.8 x 10(7) M-1 s-1) and is very stable (Kd = 6 pM) at 10 degrees C, 10 mM MOPS, pH 6.7. Charge interactions stabilize the complex by 1.4 kcal mol-1. The charge effect enters in the refolding reaction: increasing the salt concentration reduces kon dramatically and has little effect on koff. Urea and GdmCl destabilize the complex by decreasing kon and increasing koff. The slopes (m-values) of plots of ln Kd vs [cosolvent] are 0.75 +/- 0.04 and 2.8 +/- 0.3 kcal mol-1 M-1 for urea and GdmCl, respectively. The ratio mon/(mon + moff) is 0.54 +/- 0.04 for urea and 0.57 +/- 0.1 for GdmCl, where mon is the m-value for kon and moff is the m-value for koff, indicating that more than half of the sites for interaction with either cosolvent are buried in the ensemble of structures present at the transition state.
View details for Web of Science ID 000072299900052
View details for PubMedID 9485404
The pH 4 folding intermediate of apomyoglobin exists in two forms (Ia, Ib) at equilibrium. Their ratio depends on pH, urea concentration and the presence or absence of a stabilizing anion (citrate, sulfate), and it does not depend on protein concentration. The Ia and Ib species are separated by a kinetic barrier and their interconversion can be monitored by tryptophan fluorescence in stopped-flow experiments. At pH 4.2, Ib is converted to Ia at low urea concentrations and urea unfolding gives the unfolding transition of Ia. During the refolding of native (N) apomyoglobin at pH 6, starting from the acid unfolded species (U), both Ia and Ib appear as transient intermediates and both Ia and Ib appear as transient intermediates in the acid-induced unfolding of N. The results are consistent with a linear folding and unfolding pathway: U reversible Ia reversible Ib reversible N. Apomyoglobin provides the opportunity to investigate at equilibrium the structures and properties of two different kinetic folding intermediates. A non-obligatory dimeric species of the pH 4 intermediate is formed slowly and contributes to the refolding kinetics at concentrations above 5 microM. The dimer dissociates slowly and during refolding at pH 6 it forms N in a later time range than does the monomer.
View details for Web of Science ID 000072215700016
View details for PubMedID 9512718
The apomyoglobin pH 4 folding intermediate contains the A, G, and H helices of myoglobin. Helix destabilizing mutations in the A and G helices are used to test whether the pH 4 folding intermediate of apomyoglobin folds cooperatively. Single glycine or proline mutations destabilize the intermediate substantially, showing that intrinsic helix propensities are important for stability of the intermediate. The A and G helices interact to stabilize each other, as shown by the effect of mutations in the G helix on the unfolding of the A helix, which can be monitored by tryptophan fluorescence. Wild type and the most stable mutant unfold in a two-state reaction, as shown by superposition of the unfolding curves measured by two probes (far-UV circular dichroism and Trp fluorescence), while unfolding of the less stable mutants is more complex. Cooperativity and stability of folding are linked also when stabilizing anions (sulphate, perchlorate) are used to adjust stability.
View details for Web of Science ID A1997YE15900017
View details for PubMedID 9360609
Circular dichroism and NH exchange are compared directly as techniques for measuring helix content in peptides and the parameters of the helix-coil transition. To cover a broad range of helix contents, alanine-based peptides with chain lengths varying from 12 to 22 residues are examined over the temperature range from 0.6 to 26.9 degrees C in 1 M sodium chloride, 2H2O. Helix-coil transition theory independently fits both circular dichroism and exchange data, but the helix contents measured by exchange are larger than those measured by circular dichroism. The two techniques are brought into agreement by removing the assumption that the intrinsic chemical exchange rate in the helix is the same as the exchange rate measured for short unstructured model peptides. This modification allows the circular dichroism and NH exchange data to be described by the same set of helix parameters and indicates that the intrinsic exchange rate in the presence of helical structure is reduced approximately 17% relative to the rates measured in unstructured models. To test the possibility that this effect is electrostatic in origin, the sensitivity of the exchange reaction to ionic strength is determined. A substantial dependence of exchange rate on ionic strength is found, but the form of the dependence is complex. In studies of the exchange rates of native proteins, the exchange-competent form of the protein is assumed to exchange with the same rate constant as a blocked dipeptide with the identical amino acid sequences. Our result suggests that this assumption will be seriously in error in some cases because of charge effects in the protein.
View details for Web of Science ID A1997XK85600002
View details for PubMedID 9214287
To establish a framework for extrapolating the helix-forming properties of peptides from TFE/H2O mixtures (TFE = 2,2, 2-trifluoroethanol) back to water, the thermal unfolding curves have been measured by circular dichroism for four repeating-sequence peptides, with chain lengths from 7 to 22 residues. The unfolding curves were measured between 0 and 50 volume percent TFE and were fitted to the modified Lifson-Roig theory. A single set of helix-coil parameters fits the results for the four peptides at each TFE concentration; only two of the basic helix-coil parameters,
View details for Web of Science ID A1997XK09400027
View details for PubMedID 9204889
The effect on helix stability of placing a single pair of His-Asp or Asp-His residues, spaced (i, i + 3), (i, i + 4), or (i, i + 5), in an alanine-based peptide has been determined. The peptides have identical amino acid compositions, intrinsic helix propensities, and closely similar charge-helix dipole interactions, but they have different side chain interactions. Their helix contents are measured by circular dichroism over the pH range of 2-9, and the strength of a particular side chain interaction is determined from the increase in helix content over the reference peptide with the (i, i + 5) spacing. Side chain interactions are found for both the (i, i + 3) and (i, i + 4) spacings but only in the His-Asp orientation. Charged hydrogen-bond interactions occur at extreme pH values, and they are almost as strong as the ion-pair interactions at pH 5.5; but only the (i, i + 4) His-Asp peptide forms a strong hydrogen bond at pH 2, and only the (i, i + 3) peptide forms a strong hydrogen bond at pH 8.5. The ion-pair interactions are not screened effectively by 1 M NaCl, and hydrogen bonds probably account for most of their strength.
View details for Web of Science ID A1997WK47900003
View details for PubMedID 9047293
After the recent discovery of a ribonuclease A unfolding intermediate [Kiefhaber, T., et al. (1995) Nature 375, 513-515], we investigated the unfolding pathway of hen egg white lysozyme. At pH* 4.00 with D2O at 10 degrees C and 6 M guanidinium chloride, unfolding shows a single, slow kinetic phase, with a relaxation time of 3300 s when monitored by circular dichroism (CD). Exchange of the tryptophan indole nitrogen protons shows that buried Trp residues 123, 111, and 108 lose tight packing and become solvent-exposed simultaneously, with a mean relaxation time of 3300 s, similar to the CD-monitored unfolding rate. Unfolding monitored by Trp fluorescence shows, moreover, that 90% of the amplitude change occurs in a slow phase, with a relaxation time of 2400 s. Faster-unfolding phases with minor amplitudes are detected by Trp indole hydrogen exchange and by fluorescence. It is likely that these changes are caused by Trp 62 and Trp 63, active site residues which are not buried in the hydrophobic core. Lysozyme unfolding was further monitored by the histidine 15 C epsilon1 proton, which gives resolved lines for the native and unfolded species in one-dimensional 1H-NMR spectra. The majority of the unfolding reaction, 70%, occurs in a slow phase with a relaxation time of 3600 s, but there is also a rapid unfolding phase; 30% of the His 15 C epsilon1 proton resonance intensity is found at the unfolded chemical shift within tens of seconds after the start of unfolding. The amplitude of the rapid unfolding phase increases proportionally with the concentration of GdmCl denaturant present. These results show that a partially buried residue of lysozyme, histidine 15, takes part in forming an unfolding intermediate similar to the one observed earlier for valine 63 in ribonuclease A. The tryptophan side chains buried in the hydrophobic core of lysoyzme, in contrast, do not participate in forming the unfolding intermediate, as judged by proton chemical shifts. The buried tryptophan residues of dihydrofolate reductase, monitored by 19F-NMR, do participate in forming an unfolding intermediate [Hoeltzli, S. D., & Frieden, C. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9318-9322]; the difference between that study and ours may reside in the greater sensitivity of 19F to the detection of motional differences.
View details for Web of Science ID A1997WH05000034
View details for PubMedID 9063898
The helix propagation and N-cap propensities of the amino acids have been measured in alanine-based peptides in 40 volume percent trifluoroethanol (40% TFE) to determine if this helix-stabilizing solvent uniformly affects all amino acids. The propensities in 40% TFE are compared with revised values of the helix parameters of alanine-based peptides in water. Revision of the propensities in water is the result of redefining the capping statistical weights and evaluating the helix nucleation constant with N-capping explicitly included in the helix-coil model. The propagation propensities of all amino acids increase in 40% TFE relative to water, but the increases are highly variable. In water, all beta-branched and beta-substituted amino acids are helix breakers. In 40% TFE, the propagation propensities of the nonpolar amino acids increase greatly, leaving charged and neutral polar, beta-substituted amino acids as helix breakers. Glycine and proline are strong helix breakers in both solvents. Free energy differences for helix propagation (delta delta G) between alanine and other nonpolar amino acids are twice as large in water as predicted from side-chain conformational entropies, but delta delta G values in 40% TFE are close to those predicted from side-chain entropies. This dependence of delta delta G on the solvent points to a specific role of water in determining the relative helix propensities of the nonpolar amino acids. The N-cap propensities converge toward a common value in 40% TFE, suggesting that differential solvation by water contributes to the diversity of N-cap values shown by the amino acids.
View details for Web of Science ID A1996WA11500025
View details for PubMedID 8976571
Model compound studies in the literature show how Hofmeister ion interactions affect protein stability. Although model compound results are typically obtained as salting-out constants, they can be used to find out how the interactions affect protein stability. The null point in the Hofmeister series, which divides protein denaturants from stabilizers, arises from opposite interactions with different classes of groups: Hofmeister ions salt out nonpolar groups and salt in the peptide group. Theories of how Hofmeister ion interactions work need to begin by explaining the mechanisms of these two classes of interactions. Salting-out nonpolar groups has been explained by the cavity model, but its use is controversial. When applied to model compound data, the cavity model 1) uses surface tension increments to predict the observed values of the salting-out constants, within a factor of 3, and 2) predicts that the salting-out constant should increase with the number of carbon atoms in the aliphatic side chain of an amino acid, as observed. The mechanism of interaction between Hofmeister ions and the peptide group is not well understood, and it is controversial whether this interaction is ion-specific, or whether it is nonspecific and the apparent specificity resides in interactions with nearby nonpolar groups. A nonspecific salting-in interaction is known to occur between simple ions and dipolar molecules; it depends on ionic strength, not on position in the Hofmeister series. A theory by Kirkwood predicts the strength of this interaction and indicates that it depends on the first power of the ionic strength. Ions interact with proteins in various ways besides the Hofmeister ion interactions discussed here, especially by charge interactions. Much of what is known about these interactions comes from studies by Serge Timasheff and his co-workers. A general model, suitable for analyzing diverse ion-protein interactions, is provided by the two-domain model of Record and co-workers.
View details for Web of Science ID A1996VK29500037
View details for PubMedID 8889180
Little is known about the kinetic process in which stable intermediates in protein folding are formed: whether their folding is highly cooperative (two-state) or weakly cooperative is controversial. We report here that the folding and unfolding kinetics of the pH 4-stable intermediate (I1) of apomyoglobin are measurable, in the millisecond time range, when monitored by stopped-flow measurements of tryptophan fluorescence. The kinetics confirm that folding of I1 is strongly cooperative, but there is a burst phase (missing amplitude) in unfolding. If the faster steps in unfolding of I1 can be measured directly by suitable fast-reaction methods, they will give information about the nature of the folding transition.
View details for Web of Science ID A1996UV28000013
View details for PubMedID 8673605
The contribution of specific packing to the stability of the sperm whale apomyoglobin intermediate has been studied by urea denaturation monitored by circular dichroism and fluorescence. Mutations disrupting native packing sites within the subdomain formed by the A, G and H helices destabilize the intermediate, in contrast to the conclusion drawn from earlier studies of pH-induced unfolding. Based on these results, the intermediate is proposed to be stabilized by both partially formed native-like tertiary, and non-specific hydrophobic interactions forming a subdomain folding intermediate. The results help to explain how the intermediate acquires its structure and stability.
View details for Web of Science ID A1996UK24700011
View details for PubMedID 8612074
Recently, when the kinetic unfolding process of ribonuclease A was monitored by hydrogen exchange (T. Kiefhaber and R.L. Baldwin, Proc. Natl. Acad. Sci. USA, 92 (1995) 2657-2661), all peptide hydrogen bonds were found to undergo rapid exchange in a single kinetic step under conditions where unfolding is slow and the intrinsic rate of hydrogen exchange is fast (pH 8.0, 10 degrees C, 4.5 M guanidinium chloride). Comparison with the unfolding rate measured by circular dichroism indicates that hydrogen exchange is caused by the rate-limiting step of unfolding. No evidence was found for partly unfolded intermediates that are formed slowly enough to be observed by EX1 (unfolding-limited) hydrogen exchange. Some peptide NH protons were found to show, in addition to EX1 exchange, faster EX2 exchange that is base-catalyzed. The EX2 exchange is caused by species that equilibrate rapidly with the native protein at the start of the unfolding process. These species might include rapidly formed unfolding intermediates. We show here that any such unfolding intermediates must have large protection factors because the EX2 reactions of ribonuclease A under these unfolding conditions have protection factors > or = 2500.
View details for Web of Science ID A1996UK31900013
View details for PubMedID 8672722
Helix formation in a 17-residue alanine-lysine peptide and analogous peptides with specific lysine --> X substitutions, where X is 2,3-diamino-L-propionic acid, 2, 4-diamino-L-butyric acid or L-ornithine, have been examined using circular dichroism measurements. The dependence of helix content on X, its position in the sequence, and the number of lysine --> X substitutions are reasonably well described by using the Lifson-Roig theory modified to include N-capping, without explicitly considering charge-helix dipole interactions. The helix propensities for these basic amino acids increase with the length of the side-chain in the rank order 2,3-diamino-L-propionic acid < 2,4-diamino-L-butyric acid < ornithine < lysine. This parallels the increase in helix propensities with side-chain length of other polar and charged amino acids.
View details for Web of Science ID A1996UC77300022
View details for PubMedID 8648636
When NMR hydrogen exchange was used previously to monitor the kinetics of RNase A unfolding, some peptide NH protons were found to show EX2 exchange (detected by base catalysis) in addition to the expected EX1 exchange, whose rate is limited by the kinetic unfolding process. In earlier work, two groups showed independently that a restricted two-process model successfully fits published hydrogen exchange rates of native RNase A in the range 0-0.7 M guanidinium chloride. We find that this model predicts properties that are very different from the observed properties of the EX2 exchange reactions of RNase A in conditions where guanidine-induced unfolding takes place. The model predicts that EX2 exchange should be too fast to measure by the technique used, whereas it is readily measurable. Possible explanations for the contradiction are considered here, and we show that removing the restriction from the earlier two-process model is sufficient to resolve the contradiction; instead of specifying that exchange caused by global unfolding occurs by the EX2 mechanism, we allow it to occur by the general mechanism, which includes both the EX1 and EX2 cases. It is logical to remove this restriction because global unfolding of RNase A is known to give rise to EX1 exchange in these unfolding conditions. Resolving the contradiction makes it possible to determine whether populated unfolding intermediates contribute to the EX2 exchange, and this question is considered elsewhere. The results and simulations indicate that moderate or high denaturant concentrations readily give rise to EX1 exchange in native proteins. Earlier studies showed that hydrogen exchange in native proteins typically occurs by the EX2 mechanism but that high temperatures or pH values above 7 may give rise to EX1 exchange. High denaturant concentrations should be added to the list of variables likely to cause EX1 exchange.
View details for Web of Science ID A1996TY96400046
View details for PubMedID 8700871
Rapidly formed molten globule intermediates accumulate at the start of the folding reactions of several small proteins. Opinion is sharply divided as to whether they are on-pathway or off-pathway intermediates. I discuss recent experiments aimed at resolving this issue. Specific points include whether a 'rollover' in the plot of folding rate versus denaturant concentration implies that a folding intermediate is or is not on-pathway; whether the failure to observe folding intermediates for some small proteins implies a different folding mechanism or only that the intermediates are less stable; possible interpretation of 'fast-track' folding of hen lysozyme; and the significance of recent results in the search for unfolding intermediates.
View details for Web of Science ID A1996WC40200003
View details for PubMedID 9079355
Whether hydrogen bonds between side chains are energetically significant in proteins and peptides has been controversial. A method is given here for measuring these interactions in peptide helices by comparing the helix contents of peptides with 1, 2, or 3 interactions. Results are given for the glutamine--aspartate (i, i + 4) hydrogen-bond interaction. The Gibbs energy of the interaction is -1.0 kcal/mol when aspartate is charged and -0.4(4) kcal/mol when it is protonated. Magnetic resonance experiments show that the aspartate carboxylate group interacts specifically with the trans amide proton (HE) of glutamine. The interaction is observed only when the glutamine residue is N-terminal to the aspartate and when the spacing is (i, i + 4). The same stereochemistry is found in protein structures, where the (i, i + 4) glutamine-aspartate interaction occurs much more frequently than other possible arrangements.
View details for Web of Science ID A1995TB58400001
View details for PubMedID 7577910
During acid-induced unfolding of apomyoglobin, a partly folded form is observed at pH values of around four. In this form, the A, G and H helices are folded, while the rest of the molecule, including the B helix, demonstrates little structure. The partly folded form has been described as a molten globule form. To determine the factors that govern the structure and stability of this form, we introduced two helix-stabilizing mutations into the B helix, and tested their effect on the structure and stability of both the native form and the molten globule form. We show that the two Gly-->Ala replacements in the B helix produce altered fluorescence and CD properties of the partly folded intermediate, a result which implies that the B helix has become part of the structured region of the molten globule form. The helix content of a model peptide containing the sequence of the B helix is increased by the G-->A replacements, as is the helix content of the molten globule intermediate, whereas the stability and the helix content of the native protein are not altered. The observed increase in helicity is larger in the folding intermediate than in the model peptide, suggesting that nonspecific interactions, such as the hydrophobic interactions exhibited by the entire polypeptide chain, amplify the effect of intrinsic helix stability. The overall results suggest that the intrinsic stability of each individual helix is a factor in deciding whether or not that helix becomes part of the structured molten globule.
View details for Web of Science ID A1995RT96500012
View details for PubMedID 7666424
We have determined the N- and C-capping preferences of all 20 amino acids by substituting residue X in the peptides NH2-XAKAAAAKAAAAKAAGY-CONH2 and in Ac-YGAAKAAAAKAAAAKAX-CO2H. Helix contents were measured by CD spectroscopy to obtain rank orders of capping preferences. The data were further analyzed by our modified Lifson-Roig helix-coil theory, which includes capping parameters (n and c), to find free energies of capping (-RT ln n and -RT ln c), relative to Ala. Results were obtained for charged and uncharged termini and for different charged states of titratable side chains. N-cap preferences varied from Asn (best) to Gln (worst). We find, as expected, that amino acids that can accept hydrogen bonds from otherwise free backbone NH groups, such as Asn, Asp, Ser, Thr, and Cys generally have the highest N-cap preference. Gly and acetyl group are favored, as are negative charges in side chains and at the N-terminus. Our N-cap preference scale agrees well with preferences in proteins. In contrast, we find little variation when changing the identity of the C-cap residue. We find no preference for Gly at the C-cap in contrast to the situation in proteins. Both N-cap and C-cap results for Tyr and Trp are inaccurate because their aromatic groups affect the CD spectrum. The data presented here are of value in rationalizing mutations at capping sites in proteins and in predicting the helix contents of peptides.
View details for Web of Science ID A1995RG47500008
View details for PubMedID 7670375
It is commonly believed that there are no detectable intermediates in the kinetic unfolding reactions of small proteins. If such intermediates could be found, they would give important information about the nature of the transition state for unfolding, which is thought to occur close to the native state. We report here that one-dimensional proton magnetic resonance spectra recorded during the unfolding of ribonuclease A provide direct evidence for at least one unfolding intermediate in which side chains are free to rotate. This intermediate appears to be a 'dry molten globule' of the kind hypothesized by Shakhnovich and Finkelstein.
View details for Web of Science ID A1995RC18800055
View details for PubMedID 7777063
Apomyoglobin folding proceeds through a molten globule intermediate (low-salt form; I1) that has been characterized by equilibrium (pH 4) and kinetic (pH 6) folding experiments. Of the eight alpha-helices in myoglobin, three (A, G, and H) are structured in I1, while the rest appear to be unfolded. Here we report on the structure and stability of a second intermediate, the trichloroacetate form of the molten globule intermediate (I2), which is induced either from the acid-unfolded protein or from I1 by > or = 5 mM sodium trichloroacetate. Circular dichroism measurements monitoring urea- and acid-induced unfolding indicate that I2 is more highly structured and more stable than I1. Although I2 exhibits properties closer to those of the native protein, one-dimensional NMR spectra show that it maintains the lack of fixed side-chain structure that is the hallmark of a molten globule. Amide proton exchange and 1H-15N two-dimensional NMR experiments are used to identify the source of the extra helicity observed in I2. The results reveal that the existing A, G, and H helices present in I1 have become more stable in I2 and that a fourth helix--the B helix--has been incorporated into the molten globule. Available evidence is consistent with I2 being an on-pathway intermediate. The data support the view that apomyoglobin folds in a sequential fashion through a single pathway populated by intermediates of increasing structure and stability.
View details for Web of Science ID A1995RB80400039
View details for PubMedID 7777528
The factors controlling alpha-helix formation in water by peptides of defined sequence are beginning to be understood. The field is close to the point where the extent of helix formation can be predicted for peptides of any sequence. Our own approach to the problem, and the main results obtained by following this approach, are summarized below. The chief reason for studying alpha-helix formation by peptides is to understand precisely and in detail one part of the protein folding problem. Questions about peptide helix formation can be answered at a fundamental level, in terms of the physico-chemical mechanisms involved.
View details for Web of Science ID A1995RE50000010
View details for PubMedID 7632873
A previous study of the folding pathway of the major unfolded species of ribonuclease A by pulsed hydrogen exchange [Udgaonkar, J. B., & Baldwin, R. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8197-8201] showed that there is a major early folding intermediate (Il) that resembles a molten globule species in having stable secondary structure while lacking buried tyrosine side chains. Earlier work showed that there is also a late native-like folding intermediate (IN) that can bind the specific inhibitor 2'CMP and that has buried tyrosine side chains. Results are reported here indicating that Il has a well-developed tertiary structure even though its tyrosine side chains are not buried. First, optical stopped-flow experiments suggest that Il binds 2'CMP. Second, the protection against hydrogen exchange is similar in Il and IN for almost all protected amide protons studied. Third, analysis of the mechanism of hydrogen exchange in Il confirms the large protection factors reported earlier for probes in the beta-sheet of ribonuclease A and indicates that the beta-sheet is formed in Il. Other experiments are also reported that test the interpretation of pulsed hydrogen exchange studies of the folding pathway of ribonuclease A.
View details for Web of Science ID A1995QP97100027
View details for PubMedID 7696273
A sensitive test for kinetic unfolding intermediates in ribonuclease A (EC 18.104.22.168) is performed under conditions where the enzyme unfolds slowly (10 degrees C, pH 8.0, 4.5 M guanidinium chloride). Exchange of peptide NH protons (2H-1H) is used to monitor structural opening of individual hydrogen bonds during unfolding, and kinetic models are developed for hydrogen exchange during the process of protein unfolding. The analysis indicates that the kinetic process of unfolding can be monitored by EX1 exchange (limited by the rate of opening) for ribonuclease A in these conditions. Of the 49 protons whose unfolding/exchange kinetics was measured, 47 have known hydrogen bond acceptor groups. To test whether exchange during unfolding follows the EX2 (base-catalyzed) or the EX1 (uncatalyzed) mechanism, unfolding/exchange was measured both at pH 8.0 and at pH 9.0. A few faster-exchanging protons were found that undergo exchange by both EX1 and EX2 processes, but the 43 slower-exchanging protons at pH 8 undergo exchange only by the EX1 mechanism, and they have closely similar rates. Thus, it is likely that all 49 protons undergo EX1 exchange at the same rate. The results indicate that a single rate-limiting step in unfolding breaks the entire network of peptide hydrogen bonds and causes the overall unfolding of ribonuclease A. The additional exchange observed for some protons that follows the EX2 mechanism probably results from equilibrium unfolding intermediates and will be discussed elsewhere.
View details for Web of Science ID A1995QP88900051
View details for PubMedID 7708700
Pulsed hydrogen exchange and other studies of the kinetic refolding pathways of several small proteins have established that folding intermediates with native-like secondary structures are well populated, but these studies have also shown that the folding kinetics are not well synchronized. Older studies of the kinetics of formation of the native protein, monitored by optical probes, indicate that the folding kinetics should be synchronized. The model commonly used in these studies is the simple sequential model, which postulates a unique folding pathway with defined and sequential intermediates. Theories of the folding process and Monte Carlo simulations of folding suggest that neither the folding pathway nor the set of folding intermediates is unique, and that folding intermediates accumulate because of kinetic traps caused by partial misfolding. Recent experiments with cytochrome c lend support to this 'new view' of folding pathways. These different views of the folding process are discussed. Misfolding and consequent slowing down of the folding process as a result of cis-trans isomerization about prolyl peptide bonds in the unfolded protein are well known; isomerization occurs before refolding is initiated. The occurrence of equilibrium intermediates on the kinetic folding pathways of some proteins, such as alpha-lactalbumin and apomyoglobin, argues that these intermediates are not caused by kinetic traps but rather are stable intermediates under certain conditions, and this conclusion is consistent with a sequential model of folding. Folding reactions with successive kinetic intermediates, in which late intermediates are more highly folded than early intermediates, indicate that folding is hierarchical. New experiments that test the predictions of the classical and the new views are needed.
View details for Web of Science ID A1995QK89500001
View details for PubMedID 7703696
To provide a model system for understanding how the unfolding of protein alpha-helices by urea contributes to protein denaturation, urea unfolding was measured for a homologous series of helical peptides with the repeating sequence Ala-Glu-Ala-Ala-Lys-Ala and chain lengths varying from 14 to 50 residues. The dependence of the helix propagation parameter of the Zimm-Bragg model for helix-coil transition theory (s) on urea molarity ([urea]) was determined at 0 degree C with data for the entire set of peptides, and a linear dependence of In s on [urea] was found. The results were fitted by the binding-site model and by the solvent-exchange model for the interaction of urea with the peptides. Each of these thermodynamic models is able to describe the data quite well and we are not able to discern any difference between the ability of each model to fit the data. Thus a linear relation, ln s = ln s0 - (m/RT).[urea], fits the data for alpha-helix unfolding, just as others have found for protein unfolding. When the m value determined here for alpha-helix unfolding is multiplied by the number of helical residues in partly helical protein molecules, the resulting values agree within a factor of 2 with observed m values for these proteins. This result indicates that the interaction between urea and peptide groups accounts for a major part of the denaturing action of urea on proteins, as predicted earlier by some model studies with small molecules.
View details for Web of Science ID A1995QB23800038
View details for PubMedID 7816813
Straight-chain, non-natural, nonpolar amino acids norleucine, norvaline, and alpha-amino-n-butyric acid at various spacings do not interact with themselves to stabilize helix formation in alanine-based peptides, but do interact with a Tyr spaced i, i + 4 to stabilize alanine helices, similar to the helix-stabilizing i, i + 4 Tyr-Leu and Tyr-Val interactions reported earlier (Padmanabhan S, Baldwin RL, 1994, J Mol Biol 241:706-713). Leu spaced i, i + 4 from another Leu is measurably helix-stabilizing relative to the corresponding i, i + 3 pair, but less so than for i, i + 4 Val-Leu, Ile-Leu, or Phe-Leu pairs (relative to the corresponding i, i + 3 pairs) when Leu is C-terminal to the other nonpolar amino acid. Our results indicate that limited side-chain flexibility in an alpha-helix strongly favors the interaction between 2 nonpolar residues to stabilize an isolated alpha-helix.
View details for Web of Science ID A1994PZ82400011
View details for PubMedID 7703846
A helix-stabilizing interaction between tyrosine and leucine or valine has been found in alanine-based peptide helices when the spacing is i, i + 4. Control peptides have identical compositions but an i, i + 3 spacing. This is, to our knowledge, the first report of a helix-stabilizing interaction between two non-polar side-chains in an isolated helix. The results explain why, in an earlier study, leucine was found to have a helix propensity similar to that of alanine in an alanine-based peptide, whereas later work from another laboratory and our own has shown that alanine is markedly more helix-stabilizing than leucine in alanine-based peptides. The change in helix content resulting from the i, i + 4 Tyr-Leu interaction is comparable to the changes seen for other specific interactions between pairs of side-chains, such as ion-pair or Phe.His+ interactions.
View details for Web of Science ID A1994PE38300006
View details for PubMedID 8071994
The helix propensities ("s-values") of amino acids measured using short alanine-based peptides are different, in both magnitude and rank order, from those found using random sequence copolymers of a "guest" amino acid and a (hydroxyalkyl)-L-glutamine "host". The origin of these differences is investigated here. In short alanine-based peptides containing 1-5 (hydroxybutyl)-, (hydroxypropyl)-, or (hydroxyethyl)-L-glutamines (HBQ, HPQ, and HEQ, respectively), we find the rank order of helix propensities to be Ala > HBQ > HPQ > HEQ, which is consistent with earlier results for HBQ, HPQ, and HEQ homopolymers and is attributed to helix-stabilizing hydrophobic interactions [Lotan, N., Yaron, A., & Berger, A. (1966) Biopolymers 4, 365-368]. The apparent s-values of nonpolar amino acids in a 17-residue, HBQ-based peptide cluster around 1, as they do in the host-guest studies, but in contrast to results with alanine-based peptides. The differences between the host-guest results and results obtained using alanine-based peptides may be rationalized in terms of side-chain interactions involving the hydroxyalkyl moiety.
View details for Web of Science ID A1994NX97000027
View details for PubMedID 8031795
Amide proton exchange measured by one-dimensional 15N-edited proton NMR has been used to probe helical structure in an alanine-based peptide. This study is the first report of individual peptide NH exchange rates determined in a simple, repeating sequence peptide whose helical structure can be predicted by helix-coil theory. Measured protection factors directly demonstrate that the ends of the helix are frayed. The protection factors are compared to the Lifson-Roig theory, modified to include N-capping, using known values for helix propensities and N-cap propensities. Base-catalyzed exchange rates are shown to measure the extent of hydrogen bonding of the peptide NHs, and the results are fitted by a simple model in which hydrogen bonding of the peptide NH group provides protection and no exchange occurs from the hydrogen-bonded state. Protection from acid-catalyzed exchange correlates with hydrogen bonding by both the NH and CO groups of a peptide unit: the data are fitted by a model in which exchange occurs only when both hydrogen bonds formed by a peptide unit are broken. This result indicates that acid-catalyzed exchange occurs by the O-protonation mechanism, in agreement with earlier work [Perrin & Arrhenius (1982) J. Am. Chem. Soc. 104, 6693-6696; Perrin et al. (1984) J. Am. Chem. Soc. 106, 2749-2753; Tüchsen & Woodward (1985) J. Mol. Biol. 185, 421-430].
View details for Web of Science ID A1994NU49100003
View details for PubMedID 8011641
Helix propensities of the amino acids have been measured in alanine-based peptides in the absence of helix-stabilizing side-chain interactions. Fifty-eight peptides have been studied. A modified form of the Lifson-Roig theory for the helix-coil transition, which includes helix capping (Doig AJ, Chakrabartty A, Klingler TM, Baldwin RL, 1994, Biochemistry 33:3396-3403), was used to analyze the results. Substitutions were made at various positions of homologous helical peptides. Helix-capping interactions were found to contribute to helix stability, even when the substitution site was not at the end of the peptide. Analysis of our data with the original Lifson-Roig theory, which neglects capping effects, does not produce as good a fit to the experimental data as does analysis with the modified Lifson-Roig theory. At 0 degrees C, Ala is a strong helix former, Leu and Arg are helix-indifferent, and all other amino acids are helix breakers of varying severity. Because Ala has a small side chain that cannot interact significantly with other side chains, helix formation by Ala is stabilized predominantly by the backbone ("peptide H-bonds"). The implication for protein folding is that formation of peptide H-bonds can largely offset the unfavorable entropy change caused by fixing the peptide backbone. The helix propensities of most amino acids oppose folding; consequently, the majority of isolated helices derived from proteins are unstable, unless specific side-chain interactions stabilize them.
View details for Web of Science ID A1994NH62300014
View details for PubMedID 8061613
Apomyoglobin adopts a partly folded intermediate conformation (I), sometimes referred to as a molten globule intermediate, near pH 4. To determine which histidine residues trigger this partial unfolding reaction, we made mutants in which nine of the twelve histidine residues in the protein are substituted individually. We then measured acid and urea-induced unfolding curves for these substituted proteins. Two acid unfolding transitions are observed: native (N) to intermediate (I), and I to unfolded (U). These data were fitted using a simple three-state model which has been shown to give an adequate description of acid and urea-induced unfolding of wild-type apomyoglobin. The aim is to quantify changes in the apparent standard Gibbs energy differences between N, I and U, as well as the unfolding mechanism, that result from these substitutions, and to test how well the model fits data for substituted proteins. In most cases, the model fits the data reasonably well, and significant changes in fitted unfolding parameters of various mutants are also clearly visible in the primary data. The following conclusions are drawn. (1) Histidines 24 and 119 synergistically stabilize native apomyoglobin (N) at pH 8, but together destabilize N as pH is decreased below seven. (2) Histidine 36 stabilizes N when it is protonated. (3) Histidine substitutions in the heme-binding pocket (residues 64, 93 and 97) have little effect on the stability of N, suggesting that the heme-binding pocket is open. (4) Histidine substitutions affect the N to I transition but have little effect on the I to U transition. (5) The simple model we use to describe the unfolding of apomyoglobin cannot account for all the data, particularly the effects of the H36Q mutation. The effect of protonated histidine 36 on stabilizing N is not included in the model. We suggest that breaking the hydrogen bond between histidines 24 and 119 by protonation when the pH is decreased from 6 to 4 is an important part of triggering the partial unfolding of N to I, and likewise that formation of the hydrogen bond between histidines 24 and 119 may be a rate-determining step in the kinetic process of forming N from I during refolding.
View details for Web of Science ID A1994NF66000005
View details for PubMedID 8158639
We have previously shown that varying the N-terminal amino acid in alpha-helical peptides can cause large variations in helix content (Chakrabartty et al., 1993a). The Lifson-Roig theory for the helix-coil transition predicts, however, that substitutions at the N-terminus in an unacetylated peptide should have no effect on alpha-helix stability. We have therefore modified the theory to include these N-capping effects by assigning a statistical weight (the "n-value") to the amino acid immediately preceding a stretch of helical residues. The n-value measures the N-capping propensity of an amino acid, and like the helix propensity (w-value), it is independent of neighboring residues or positions in sequence. The new theory was used, with the experimental data for these substitutions, to calculate n-values and, hence, free energies for N-capping for the amino acids Gln, Ala, Val, Met, Pro, Ile, Leu, Thr, Gly, Ser, and Asn as well as for the acetyl group, which is commonly used to cap peptides. The free energies vary by approximately 1 kcal mol-1 from Gln (worst) to Asn (best), and the acetyl group is nearly as effective as Asn. N-Capping free energies were also found for Leu, Thr, Gly, Ser, and Asn when the N-terminus is charged at pH 5. The unfavorable effect of protonation of the N-terminus in an alpha-helix was found to be approximately 0.5 kcal mol-1. Our results agree well with a survey of N-capping preferences from protein crystal structures and are compared to results from site-directed mutagenesis of N-caps in proteins.
View details for Web of Science ID A1994NC04100033
View details for PubMedID 8136377
Helix content of peptides with various uncharged nonaromatic amino acids at either the N-terminal or C-terminal position has been determined. The choice of N-terminal amino acid has a major effect on helix stability: asparagine is the best, glycine is very good, and glutamine is the worst helix-stabilizing amino acid at this position. The rank order of helix stabilization parallels the frequencies of these amino acids at the N-terminal boundary (N-cap) position of helices in proteins found by Richardson and Richardson [Richardson, J. S. & Richardson, D. C. (1988) Science 240, 1648-1652], and the N-terminal amino acid in a peptide composed of helix-forming amino acids may be considered as the N-cap residue. The choice of C-terminal amino acid has only a minor effect on helix stability. N-capping interactions may be responsible for the asymmetric distribution of helix content within a given peptide found by various workers. An acetyl group on the N-terminal alpha-amino function cancels the N-cap effect and the acetyl group is equivalent to N-terminal asparagine in an unacetylated peptide. Our results demonstrate a close relationship between the mechanisms of alpha-helix formation in peptides and in proteins.
View details for Web of Science ID A1993MK09400092
View details for PubMedID 8248248
To determine whether a charged histidine side chain affects alpha-helix stability only when histidine is close to one end of the helix or also when it is in the central region, we substitute a single histidine residue at many positions in two reference peptides and measure helix stability and histidine pKa. The position of a charged histidine residue has a major effect on helix stability in 0.01 M NaCl: the helix content of a 17-residue peptide is 24% when histidine is at position 3 compared to 76% when it is at position 17. This dependence of helix content on histidine position decreases sharply in 1 M NaCl, as expected for counterion screening of the charge-helix dipole interaction. Results at interior positions indicate that the position of a charged histidine residue affects helix stability at these positions. Unexpectedly high values of the helix content are found when either neutral or charged histidine is at one of the last three C-terminal positions, suggesting that either form can stabilize an isolated helix by hydrogen bonding to a main-chain CO group.
View details for Web of Science ID A1993MK09400093
View details for PubMedID 8248249
Amide (NH) proton exchange rates were measured in 0.0 to 0.7 M guanidinium chloride (GdmCl) for 23 slowly exchanging peptide NH protons of ribonuclease A (RNase A) at pH* 5.5 (uncorrected pH measured in D2O), 34 degrees C. The purpose was to find out whether GdmCl induces exchange through binding to exchange intermediates that are partly or wholly unfolded. It was predicted that, when the logarithm of the exchange rate is plotted as a function of the molarity of GdmCl, the slope should be a measure of the amount of buried surface area exposed to GdmCl in the exchange intermediate. The results indicate that these concentrations of GdmCl do induce exchange by means of a partial unfolding mechanism for all 23 protons; this implies that exchange reactions can be used to study the unfolding and stability of local regions. Of the 23 protons, nine also show a second mechanism of exchange at lower concentrations of GdmCl, a mechanism that is nearly independent of GdmCl concentration and is termed "limited structural fluctuation."
View details for Web of Science ID A1993MF43800027
View details for PubMedID 8235609
View details for Web of Science ID A1993MD05800013
A single aspartate residue has been placed at various positions in individual peptides for which the alanine-based reference peptide is electrically neutral, and the helix contents of the peptides have been measured by circular dichroism. The dependence of peptide helix content on aspartate position has been used to determine the helix propensity (s-value). Both the charged (Asp-) and uncharged (Asp0) forms of the aspartate residue are strong helix breakers and have identical s-values of 0.29 at 0 degree C. The interaction of Asp- with the helix dipole affects helix stability at positions throughout the helix, not only near the N-terminus, where the interaction is helix stabilizing, and the C-terminus, where it is destabilizing. Comparison of the helix contents at acidic pH (Asp0) and at neutral pH (Asp-) shows that the charge-helix dipole interaction is screened slowly with increasing NaCl concentration, and screening is not complete even at 4.8 M NaCl. Lastly, a helix-stabilizing hydrogen-bond interaction between glutamine and aspartate (spacing i, i + 4) has been found. This side-chain interaction is specific for both the orientation and spacing of the glutamine and aspartate residues and is resistant to screening by NaCl.
View details for Web of Science ID A1993MA68800006
View details for PubMedID 8251935
A single pair of Glu and Lys residues has been placed at four different spacings, and in both orientations, in an otherwise neutral alanine-glutamine peptide helix, and the contribution to helix stability of the different Glu-Lys interactions has been measured. The contribution from the interaction of each charged side chain with the helix macrodipole has also been determined. A side-chain interaction between Gln and Glu, when the spacing is (i,i+4), has been detected and quantified. The interactions have been divided into contributions from hydrogen bonds (independent of the concentration of NaCl) and from electrostatic interactions (present in 10 mM NaCl, absent in 2.5 M NaCl). The major results are as follows: (1) The (i,i+3) and (i,i+4) Glu-Lys interactions are helix-stabilizing and are similar in strength to each other, regardless of the orientation of the side chains. (2) Hydrogen bonds provide the major contribution to these side-chain interactions, as shown by the following facts. First, the major part of the interaction observed in 10 mM NaCl, pH 7, is still present in 2.5 M NaCl. Second, the interaction found at pH 2 is equally as strong as that found in 2.5 M NaCl at pH 7. (3) The (i,i+4) Gln-Glu side-chain hydrogen bond is as strong as the hydrogen-bond component of the Glu-Lys interaction at both pH 2 and pH 7. The Gln-Glu interaction differs from the Glu-Lys interaction in being specific both for the orientation and the spacing of the residues. (4) No significant hydrogen-bonding interaction was found for the (i,i+1) or (i, i+2) Glu-Lys spacings, either at pH 2 or at pH 7, in 2.5 M NaCl. At 10 mM NaCl and pH 7, these spacings show a helix-destabilizing electrostatic interaction which probably results from stabilization of the coil conformation.
View details for Web of Science ID A1993LY29400019
View details for PubMedID 8373771
Peptides of the sequence Ac-XKAAAAKAAAAKAAAAK-amide, where X is Tyr, Trp, or Ala, produce circular dichroism spectra that are typical of the alpha-helix; there are, however, significant differences between the Tyr, Trp, or Ala peptides in the magnitudes of the far-ultraviolet bands. A tyrosine or tryptophan residue is needed in each peptide in order to measure accurately the peptide concentration and the mean residue ellipticity. The N- or C-terminal position is chosen because helix fraying is greatest at each end and the Tyr or Trp residue should influence the helix content of the peptide least at these positions. Amide proton exchange measurements by proton nuclear magnetic resonance spectroscopy indicate that the Tyr, Trp, and Ala peptides possess similar amounts of H-bonded secondary structure. Comparison of the far-ultraviolet circular dichroism and absorption spectra of these peptides suggests that the differences in circular dichroism arise in each case from an induced aromatic circular dichroism band, which is positive for Tyr and negative for Trp. Insertion of one to three Gly residues between the aromatic residue and the rest of the helical sequence reduces the induced aromatic band to insignificant levels. Using this procedure of inserting Gly residues between the Tyr and the rest of the helical sequence, we remeasured the helix propensity of Gly. We find that the Ala:Gly ratio of helix propensities is 40, as opposed to our previous estimate of 100 determined using the Tyr peptide without considering the aromatic contribution of Tyr in the analysis [Chakrabartty, A., Schellman, J. A., & Baldwin, R. L. (1991) Nature 351, 586-588].
View details for Web of Science ID A1993LF06300010
View details for PubMedID 8504077
The molten globule model for the beginning of the folding process, which originated with Kuwajima's studies of alpha-lactalbumin (Kuwajima, K., 1989, Proteins Struct. Funct. Genet. 6, 87-103, and references therein), states that, for those proteins that exhibit equilibrium molten globule intermediates, the molten globule is a major kinetic intermediate near the start of the folding pathway. Pulsed hydrogen-deuterium exchange measurements confirm this model for apomyoglobin (Jennings, P.A. & Wright, P.E., in prep.). The energetics of the acid-induced unfolding transition, which have been determined by fitting a minimal three-state model (N<-->I<-->U; N = native, I = molten globule intermediate, U = unfolded) show that I is more stable than U at neutral pH (Barrick, D. & Baldwin, R.L., 1993, Biochemistry 32, in press), which provides an explanation for why I is formed from U at the start of folding. Hydrogen exchange rates measured by two-dimensional NMR for individual peptide NH protons, taken together with the CD spectrum of I, indicate that moderately stable helices are present in I at the locations of the A, G, and H helices of native myoglobin (Hughson, F.M., Wright, P.E., & Baldwin, R.L., 1990, Science 249, 1544-1548). Directed mutagnesis experiments indicate that the interactions between the A, G, and H helices in I are loose (Hughson, F.M., Barrick, D., & Baldwin, R.L., 1991, Biochemistry 30, 4113-4118), which can explain why I is formed rapidly from U at the start of folding.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for PubMedID 8318892
Perchlorate-denatured ribonuclease A (PDR) is known to show a circular dichroism (CD) spectrum suggestive of substantial secondary structure. Thus, PDR may be a molten globule form of ribonuclease A. We find that any secondary structure present in PDR does not provide measurable protection against amide proton exchange, and PDR does not belong to the class of structured molten globules. CD spectra of short peptides show that the perchlorate anion affects these spectra in a way that could be mistaken for secondary structure formation; thus, caution must be used in interpreting CD spectra of peptides and proteins taken in perchlorate solutions.
View details for Web of Science ID A1993LA56000017
View details for PubMedID 8387338
We give a quantitative description of the urea- and acid-induced transitions of apomyoglobin at 0 degree C and 2 mM sodium citrate. Our data consist of two series of unfolding curves: (1) acid-induced unfolding carried out in the presence of various concentrations of urea and (2) urea-induced unfolding at various pH values. A three-state equation is derived which relates the stability of three different conformations of apomyoglobin (native [N], unfolded [U], and intermediate [I]) as a function of urea and of pH. This equation fits our data reasonably well. The parameters which give the best fit have both thermodynamic and structural implications for N, I, and U. Specifically, I is closer in Gibbs energy to U than to N, indicating that side-chain packing results in much of the stability of native protein structure. The equilibria between N and I and between I and U are equally sensitive to urea, suggesting that much of the surface of I is inaccessible to solvent. The acid-induced transition in which N unfolds can be described as the result of titration of approximately two histidines with low pKaS in N. Under physiological conditions (neutral pH, no urea) I is the most stable non-native conformation.
View details for Web of Science ID A1993KX17900035
View details for PubMedID 8466917
Two models have been considered for the helix-stabilizing Phe-His+ interaction in C-peptide: (1) the H-bond model in which His+ acts as an H-bond donor and the aromatic ring of Phe acts as an acceptor, and (2) a helix dipole model, in which Phe constrains His so that there is a stronger interaction between His+ and the helix dipole. To decide between these models, we compared the effect on helix stability of the Phe-His interaction near the middle versus close to the C terminus of an alanine-based peptide. The nature of the interaction is the same at either position, in agreement with the H-bond model. The results show further that a weak helix-stabilizing Phe-His interaction can be detected when His is uncharged. Replacement of Phe by the saturated analog Cha (beta-cyclohexylalanine) gives no interaction, as predicted by the H-bond model.
View details for Web of Science ID A1993KR93300025
View details for PubMedID 8450542
The helix-stabilizing effects of repeating pairs of Asp-Arg and Glu-Arg residues have been characterized using a peptide system of the same design used earlier to study Glu-Lys (Marqusee, S. & Baldwin, R.L., 1987, Proc. Natl. Acad. Sci. USA 84, 8898-8902) and Asp-Lys ion pairs (Marqusee, S. & Baldwin, R.L., 1990, In Protein Folding [Gierasch, L.M. & King, J., Eds.], pp. 85-94, AAAS, Washington, D.C.). The consequences of breaking ion pair and charge-helix dipole interactions by titration to pH 2 have been compared with the results of screening these interactions with NaCl at pH 7.0 and pH 2.5. The four peptides in each set contain three pairs of acidic (A) and basic (B) residues spaced either i, i + 4 or i, i + 3 apart. In one peptide of each kind the pairwise order of residues is AB, with the charges oriented favorably to the helix macrodipole, and in the other peptide the order is BA. The results are as follows: (1) Remarkably, both Asp-Arg and Glu-Arg peptides show the same pattern of helix stabilization at pH 7.0 found earlier for Glu-Lys and Asp-Lys peptides: i + 4 AB > i + 4 BA approximately i + 3 AB > i + 3 BA. (2) The ion pairs and charge-helix dipole interactions cannot be cleanly separated, but the results suggest that both interactions make important contributions to helix stability.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1993KG86400008
View details for PubMedID 8443591
View details for Web of Science ID A1993LJ63700013
A challenge in understanding the thermodynamics of protein unfolding is to explain the 1979 puzzle posed by Privalov. Why do values of the specific enthalpy and specific entropy of unfolding both converge to common values at approximately the same temperature (Th* approximately equal to Ts*) when extrapolated linearly versus temperature? In 1986, a liquid hydrocarbon model gave an explanation for convergence of the specific entropies at Ts*: it happens because the contribution of the hydrophobic effect to the entropy of unfolding goes to zero at Ts*. The reason for convergence of the specific enthalpies at Th* and for the equality Th* approximately equal to Ts* has remained, however, a matter for speculation; recently, some explanations have been given that are based on models for polar interactions in protein folding. We show here that the relation Th* approximately equal to Ts* can be derived straightforwardly without making any assumptions either about polar interactions or about splitting the hydrophobic interaction into two terms--one for the "hydrophobic hydration" and the other for the residual effect, as suggested recently. Thus, the liquid hydrocarbon model explains both halves of Privalov's puzzle. A similar conclusion has been reached independently by A. Doig and D. H. Williams (personal communication). It has been proposed recently that a correction should be made for the relative sizes of a hydrocarbon solute and water when computing the thermodynamic properties of the hydrophobic interaction from a solvent transfer experiment. This correction affects the temperature at which the entropy of transfer equals zero, and it is important to evaluate its effect on the convergence temperature Ts*. We show that making the size correction does not change the conclusion, reached earlier, that the liquid hydrocarbon model explains the convergence of the specific entropies of protein unfolding.
View details for Web of Science ID A1992JF85600093
View details for PubMedID 1496007
A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give delta Tm approximately equal to 10 degrees C and delta delta G0 (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis<==>trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.
View details for Web of Science ID A1992JF88300009
View details for PubMedID 1338975
Ribonuclease A is known to form an equilibrium mixture of fast-folding (UF) and slow-folding (US) species. Rapid unfolding to UF is then followed by a reaction in the unfolded state, which produces a mixture of UF, USII, USI, and possibly also minor populations of other US species. The two cis proline residues, P93 and P114, are logical candidates for producing the major US species after unfolding, by slow cis <==> trans isomerization. Much work has been done in the past on testing this proposal, but the results have been controversial. Site-directed mutagenesis is used here. Four single mutants, P93A, P93S, P114A, and P114G, and also the double mutant P93A, P114G have been made and tested for the formation of US species after unfolding. The single mutants P114G and P114A still show slow isomerization reactions after unfolding that produce US species; thus, Pro 114 is not required for the formation of at least one of the major US species of ribonuclease A. Both the refolding kinetics and the isomerization kinetics after unfolding of the Pro 93 single mutants are unexpectedly complex, possibly because the substituted amino acid forms a cis peptide bond, which should undergo cis --> trans isomerization after unfolding. The kinetics of peptide bond isomerization are not understood at present and the Pro 93 single mutants cannot be used yet to investigate the role of Pro 93 in forming the US species of ribonuclease A. The double mutant P93A, P114G shows single exponential kinetics measured by CD, and it shows no evidence of isomerization after unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1992JF88300010
View details for PubMedID 1304376
The kinetics of amide proton exchange (1H----2H) have been measured by proton nuclear magnetic resonance spectroscopy for a set of helical peptides with the generic formula Ac-(AAKAA)m Y-NH2 and with chain lengths varying from 6 to 51 residues. The integrated intensity of the amide resonances has been measured as a function of time in 2H2O at pH* 2.50. Exchange kinetics for these peptides can be modeled by applying the Lifson-Roig treatment for the helix-to-coil transition. The Lifson-Roig equation is used to compute the probability that each residue is helical, as defined by its backbone (phi, psi) angles. A recursion formula then is used to find the probability that the backbone amide proton of each residue is hydrogen bonded. The peptide helix can be treated as a homopolymer, and direct exchange from the helix can be neglected. The expression for the exchange kinetics contains only three unknown parameters: the rate constant for exchange of a non-hydrogen-bonded (random coil) backbone amide proton and the nucleation (v2) and propagation (w) parameters of the Lifson-Roig theory. The fit of the exchange curves to these three parameters is very good, and the values for v2 and w agree with those derived from circular dichroism studies of the thermally-induced unfolding of related peptides [Scholtz, J.M., Qian, H., York, E.J., Stewart, J.M., & Baldwin, R.L. (1991) Biopolymers (in press]).
View details for Web of Science ID A1992HD15700001
View details for PubMedID 1310608
Thermal unfolding curves have been measured for a series of short alanine-based peptides that contain repeating sequences and varying chain lengths. Standard helix-coil theory successfully fits the observed transition curves, even for these short peptides. The results provide values for sigma, the helix nucleation constant, delta H0, the enthalpy change on helix formation, and for s (0 degree C), the average helix propagation parameter at 0 degree C. The enthalpy change agrees with the value determined calorimetrically. The success of helix-coil theory in describing the unfolding transitions of short peptides in water indicates that helical propensities, or s values, can be determined from substitution experiments in short alanine-based peptides.
View details for Web of Science ID A1991HA43600002
View details for PubMedID 1814498
A search has been made for position effects on apparent helix propensities when another amino acid is substituted for alanine in the C-peptide helix of ribonuclease A. Three internal alanine residues (Ala4, Ala5, Ala6) are used as sites for substitution. Five amino acids, Glu, His, Arg, Lys and Phe, are substituted singly in individual peptides at each of these three positions, and the pH profiles of helix content for the substituted peptides have been determined. The effect of using an acetyl or a succinyl amino-terminal-blocking group has also been determined for each substitution. A strong position effect is found at Ala5: the helix content of the substituted peptide is significantly higher for substitution at position 5 than at positions 4 or 6 in almost all cases. The reason for the position 5 effect is unknown. The results also show that electrostatic interactions often influence substitution experiments, and they provide data on the variability of substitution experiments made with a natural sequence peptide.
View details for Web of Science ID A1991GN21000025
View details for PubMedID 1942058
Hydrogen exchange has been used to test for the presence of nonrandom structure in thermally denatured ribonuclease A (RNase A). Quenched-flow methods and 2D 1H NMR spectroscopy were used to measure exchange rates for 36 backbone amide protons (NHs) at 65 degrees C and at pH* (uncorrected pH measured in D2O) values ranging from 1.5 to 3.8. The results show that exchange is approximately that predicted for a disordered polypeptide [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158]; we thus are unable to detect any stable hydrogen-bonded structure in thermally denatured RNase A. Two observations suggest, however, that the predicted rates should be viewed with some caution. First, we discovered that one of the approximations made by Molday et al. (1972), that exchange for valine NHs is similar to that for alanine NHs, had to be modified; the exchange rates for valine NHs are about 4-fold slower. Second, the pH minima for exchange tend to fall at lower pH values than predicted, by as much as 0.45 pH units. These results are in accord with those of Roder and co-workers for bovine pancreatic trypsin inhibitor [see Table I in Roder, H., Wagner, G., & Wüthrich, K. (1985) Biochemistry 24, 7407-7411]. The origin of the disagreement between predicted and observed pH minima is unknown but may be the high net positive charge on these proteins at low pH. In common with some other thermally unfolded proteins, heat-denatured ribonuclease A shows a significant circular dichroism spectrum in the far-ultraviolet region [Labhardt, A. M. (1982) J. Mol. Biol. 157, 331-355].(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1991GK06000014
View details for PubMedID 1911782
View details for PubMedID 15336124
The standard view of alpha helix formation in water, based on helix propensities determined by the host-guest method, is that differences in helix propensity among the amino acids are small, except for proline, and that the average value of the helix propagation parameter s is near 1. A contradictory view of alpha helix formation in water is emerging from substitution experiments with short, unique-sequence peptides that contain only naturally occurring amino acids. Short peptides that contain only alanine and lysine, or alanine and glutamate, form surprisingly stable monomeric helices in water and substitution of a single alanine residue by another amino acid in these or related peptides produces a wide range of changes in helix content, depending on which amino acid is substituted for alanine. We show here that the ratio of the helix propensities of alanine to glycine is large, about 100, in substitution experiments with a 17-residue reference peptide containing alanine and lysine. The helix propensity is identified with s, the helix propagation parameter of the statistical mechanics model for alpha helix formation, and the results are interpreted by the Lifson-Roig theory. Single alanine----glycine substitutions have been made at a series of positions in individual peptides. The helix-destabilizing effect of an Ala----Gly substitution depends strongly on its position in the helix, as predicted by the Lifson-Roig theory if the ratio of s values for Ala:Gly is large.
View details for Web of Science ID A1991FR03400065
View details for PubMedID 2046766
The effect on overall alpha-helix content of substituting proline for alanine has been determined at 5 positions (1, 2, 4, 5, and 13) of a 13-residue peptide related in sequence to residues 1-13 of ribonuclease A. The helix content falls off rapidly as proline is moved inward, and the proline residue effectively truncates the helix. No helix-stabilizing effect of proline is found at positions 2 or 4 within the first turn of the helix. Proline substitution at either end position (1, 13) has little effect on overall helix content, in agreement with an earlier study of glycine for alanine substitutions. The two end residues of the helix appear to be strongly frayed.
View details for Web of Science ID A1991FQ55300026
View details for PubMedID 2043620
For comparison with earlier data on naturally occurring non-polar amino acids (Ala, Leu, Phe, Val, Ile), the comparative helix-forming tendencies have been measured for non-polar amino acid residues that have unbranched side-chains, with an ethyl, propyl or butyl group, and also for methionine. The substitutions are made in a 17-residue alanine-based peptide. The results show that straight-chain non-polar amino acids have high helix-forming tendencies compared to beta-branched non-polar amino acids. Restriction of side-chain conformations in the helix, with a corresponding reduction in conformational entropy, is the likely explanation. There is a small increase in helix-forming tendency as the side-chain increases in length from ethyl to butyl, which suggests that a helix-stabilizing hydrophobic interaction is being detected.
View details for Web of Science ID A1991FM82400001
View details for PubMedID 2038048
A partly folded form (I) of apomyoglobin has an alpha-helix content of about 35%; in an earlier study, hydrogen exchange revealed that the A, G, and H helices are folded, while much of the rest of the protein is not [Hughson, F. M., Wright, P. E., & Baldwin, R. L. (1990) Science 249, 1544-1548]. Because A, G, and H form a compact subdomain in native myoglobin, we proposed that nativelike packing interactions among the three helices might be retained in the I form of apomyoglobin. To test this proposal, disruptive mutations were introduced into the A.H and G.H helix packing sites. These mutations destabilize native apomyoglobin relative to I. In contrast, the stability of I is relatively insensitive to mutation; in particular, side-chain volume alone does not appear to be important. These results indicate that the I form is not stabilized by nativelike A.H and G.H packing interactions. In support of this we show that partly helical peptides derived from the G and H helix regions of myoglobin do not pair in solution. Since the isolated G and H peptides are at best only partly helical, some type of interaction must stabilize these helices in the I form. Small increases in the stability of I are seen when mutation introduces a side chain of increased nonpolar surface area. We suggest that I is stabilized by relatively nonspecific hydrophobic interactions that allow it to adapt easily to mutation. In this and other respects, I appears to conform to the "molten globule" model, with the caveat that only part of the polypeptide chain appears to participate in the globule.
View details for Web of Science ID A1991FK23400001
View details for PubMedID 2021603
The enthalpy change (delta H) accompanying the alpha-helix to random coil transition in water has been determined calorimetrically for a 50-residue peptide of defined sequence that contains primarily alanine. The enthalpy of helix formation is one of the basic parameters needed to predict thermal unfolding curves for peptide helices and it provides a starting point for analysis of the peptide hydrogen bond. The experimental uncertainty in delta H reflects the fact that the transition curve is too broad to measure in its entirety, which precludes fitting the baselines directly. A lower limit for delta H of unfolding, 0.9 kcal/mol per residue, is given by assuming that the change in heat capacity (delta Cp) is zero, and allowing the baseline to intersect the transition curve at the lowest measured Cp value. Use of the van't Hoff equation plus least-squares fitting to determine a more probable baseline gives delta H = 1.3 kcal/mol per residue. Earlier studies of poly(L-lysine) and poly(L-glutamate) have given 1.1 kcal/mol per residue. Those investigations, along with our present result, suggest that the side chain has little effect on delta H. The possibility that the peptide hydrogen bond shows a correspondingly large delta H, and the implications for protein stability, are discussed.
View details for Web of Science ID A1991FE86400051
View details for PubMedID 2011594
Studies of a stable molten globule intermediate (I) of apomyoglobin have shown that: (1) the A, G and H helices, but not the B and E helices, of myoglobin are stabilized in I, (2) individual peptides containing the G and H sequences do not show stable helix formation, although the H peptide shows partial (30%) helix formation, and (3) the mechanism by which the A, G and H helices are stabilized in I is not side chain interdigitation between helices at the helix contact sites of myoglobin. Consequently, the molten globule intermediate confers stability on the A, G and H helices, and the mechanism of stabilization is not the direct interaction found in native myoglobin. Kinetic studies of the folding reactions of small proteins have shown folding intermediates that could be either framework intermediates or molten globule intermediates, but a clear distinction between these two classes of kinetic intermediates has been lacking. An operational distinction is proposed here: molten globule intermediates are not stabilized by side chain interdigitation at contact sites between units of secondary structure found in the corresponding native protein, whereas framework intermediates are stabilized in this way. Site-directed mutagenesis experiments can distinguish between the two classes of intermediate. On the basis of this definition, the kinetic folding intermediates that are detected by far-UV circular dichroism can be molten globule intermediates, and when both a molten globule and a framework intermediate occur on the same folding pathway, the molten globule intermediate should precede the framework intermediate. Protection of individual amide protons against exchange has given the most detailed information thus far about the structures of folding intermediates in non-covalent folding reactions. It is possible that amide proton protection might occur during folding either by a non-specific mechanism, such as a hydrophobic collapse, or by the formation and later breakdown of non-native secondary structure; either event would pose a serious problem for interpretation of the results. Tests are available for assessing whether either event occurs, and they are discussed here.
View details for Web of Science ID A1991GP94800012
View details for PubMedID 1667633
Pulsed hydrogen exchange (2H-1H) is used to characterize the folding process of ribonuclease A (disulfide bonds intact). The results show one principal early folding intermediate (I1), which is formed rapidly after the start of folding and whose proton-exchange properties change with the time of folding. All probes that are hydrogen bonded within the beta-sheet of native ribonuclease A are protected in I1. Thus, the results suggest that the beta-sheet is formed rapidly and cooperatively. The initial protection factors of probes in the beta-sheet are between 10 and 100, but they increase with time of folding and exceed 1000 at 400 msec from the start of folding. Thus, the beta-sheet is only moderately stable when it is first formed, but subsequent events stabilize it, possibly through interactions involving hydrophobic side chains. The large protection factors of the beta-sheet probes in an early folding intermediate are unexpected and remarkable. Probes in the three alpha-helices are fewer in number and give less accurate data than the beta-strand probes. The folding kinetics expected for a simple sequential model of folding are outlined. An important difference between the observed and predicted behavior is that the early folding intermediate is not fully populated when it is first formed.
View details for Web of Science ID A1990EG22000003
View details for PubMedID 2236032
To understand why proteins adopt particular three-dimensional structures, it is important to elucidate the hierarchy of interactions that stabilize the native state. Proteins in partly folded states can be used to dissect protein organizational hierarchies. A partly folded apomyoglobin intermediate has now been characterized structurally by trapping slowly exchanging peptide NH protons and analyzing them by two-dimensional 1H-NMR (nuclear magnetic resonance). Protons in the A, G, and H helix regions are protected from exchange, while protons in the B and E helix regions exchange freely. On the basis of these results and the three-dimensional structure of native myoglobin, a structural model is presented for the partly folded intermediate in which a compact subdomain retains structure while the remainder of the protein is essentially unfolded.
View details for Web of Science ID A1990EA46300031
View details for PubMedID 2218495
Previous studies have identified Lys 1, Glu 2, and His 12 as the charged residues responsible for the pH-dependent stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A). Here we examine whether the helix-stabilizing behavior of Glu 2- results from a Glu 2- ... Arg 10+ interaction, which is known to be present in the crystal structure of ribonuclease A. The general approach is to measure the helix content of C-peptide analogs as a function of three variables: pH (titration of ionizing groups), amino acid identity (substitution test), and NaCl concentration (ion screening test). In order to interpret the results of residue replacement, several factors in addition to the putative Glu 2- ... Arg 10+ interaction have been studied: intrinsic helix-forming tendencies of amino acids; interactions of charged residues with the alpha-helix macrodipole; and helix-lengthening effects. The results provide strong evidence that the Glu 2- ... Arg 10+ interaction is linked to helix formation and contributes to the stability of the isolated C-peptide helix. NMR evidence supports these conclusions and suggests that this interaction also acts as the N-terminal helix stop signal. The implications of this work for protein folding and stability are discussed.
View details for Web of Science ID A1990EA39300012
View details for PubMedID 1981024
An important issue in understanding the relationship between protein sequence and structure is the degree to which different amino acids favour the formation of particular types of secondary structure. Estimates of the 'helix-forming tendency' of amino acids have been made based on 'host-guest' experiments, in which copolymers are made of the amino acid of interest (the 'guest') and a host residue (typically hydroxypropyl- or hydroxybutyl-L-glutamine). Recently, however, short alanine-based peptides were found to form stable monomeric helices in water, contrary to the result predicted from host-guest experiments. We have now measured the helix-forming tendency of five different nonpolar amino acids (Ala, Ile, Leu, Phe, Val) by substituting each in turn for alanine in a 17-residue alanine-based peptide and determining the extent of alpha-helix formation. Our results differ from those of host-guest experiments both in the degree of variation in helix-forming tendency of different amino acids, and in the rank order of the helix-forming tendency. We conclude that the helix-forming tendency of a particular amino acid depends on the sequence context in which it occurs; and the restriction of side-chain rotamer conformations is important in determining the helix-forming tendency.
View details for Web of Science ID A1990CU13800067
View details for PubMedID 2314462
Previous studies have demonstrated that His 12 plays a major role in the pH-dependent stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A). Here, amino acid replacement experiments show that His 12+ stabilizes the C-peptide helix chiefly by interacting with Phe 8. The Phe 8 ... His 12+ ring interaction is specific for the protonated form of His 12 (His 12+) and the interaction is not screened significantly by NaCl, unlike the charged group ... helix dipole interactions studied earlier in C-peptide. Analogs of C-peptide that are unable to form the Phe 8 ... His 12+ interaction show large increases in helix content for Phe----Ala and His----Ala. Therefore, the helical tendencies of the individual residues Phe, His, and Ala are important in determining the result of a replacement experiment. Since the side chains of Phe 8 and His 12 probably interact within the N-terminal helix of ribonuclease A, the existence of the Phe 8 ... His 12+ interaction in the isolated C-peptide helix adds to the evidence that the C-peptide helix is an autonomous folding unit.
View details for Web of Science ID A1990CP54100002
View details for PubMedID 2328280
View details for Web of Science ID A1990BR06M00008
Two-dimensional NMR experiments have been performed on a peptide, succinyl-AE-TAAAKFLRAHA-NH2, related to the amino-terminal sequence of ribonuclease A. This peptide contains 50-60% helix in 0.1 M NaCl solution, pH 5.2, 3 degrees C, as measured by circular dichroism. NOESY spectra of the peptide in aqueous solution at low temperatures show a number of NOE connectivities that are used to determine the highly populated conformations of the peptide in solution. Short-range dNN(i, i + 1) and d alpha N(i, i + 1) connectivities and medium-range d alpha beta(i, i + 3) and d alpha N(i, i + 3) connectivities are detected. The pattern of NOE connectivities unambiguously establishes the presence of helix in this peptide. The magnitudes of the 3JHN alpha coupling constants and the intensities of the dNN(i, i + 1) and d alpha N(i,i + 1) NOEs allow the evaluation of the position of the helix along the peptide backbone. These data indicate that the amino terminus of the peptide is less helical than the remainder of the peptide. The observation of several long-range NOEs that are atypical of helices indicates the presence of a high population of peptide molecules in which the first three residues are distorted out of the helical conformation. The absence of these NOEs in a related peptide, RN-31, in which Arg 10 has been changed to Ala, suggests that this distortion at the amino-terminal end of the peptide arises from the formation of a salt bridge between Glu 2 and Arg 10.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1989AN01400042
View details for PubMedID 2819049
Short, 16-residue, alanine-based peptides show stable alpha-helix formation in H2O. This result is surprising when contrasted with the classical view that regards the alpha-helix as a marginally stable structure in H2O and considers short helices unstable. The alanine-based peptides are solubilized by insertion of three or more residues of a single charge type, lysine (+) or glutamic acid (-). The results cannot be explained by helix stabilization resulting from concentration-dependent association or by the interaction of charged residues with the helix dipole. Our results are not predicted by the parameters for alanine and lysine that have been determined by the "host-guest" method: these parameters predict that a 16-residue peptide should not show measurable alpha-helix formation. Analysis of the role of the hydrophobic interaction in alpha-helix formation [Richards, F.M. & Richmond, T. (1978) in Molecular Interactions and Activity in Proteins, Ciba Foundation Symposium 60, ed. Wolstenholme, G.E. (Excepta Medica Amsterdam), pp. 23-25] does not show an unusually strong hydrophobic interaction in a helical block of alanine residues. The likely explanation for our results is, therefore, that individual alanine residues have a high helical potential. It is not yet known whether any other amino acids show this property, and the origin of this property is also unknown.
View details for Web of Science ID A1989AG35900015
View details for PubMedID 2748584
Site-directed mutagenesis has been used to study the effect on the stability of human apomyoglobin (apoMb) of modifying the size, hydrophobicity, and charge of a central residue in the G.B helix-helix packing interface. Some stability measurements have also been made on the corresponding holomyoglobins (heme present). Cys-110, a central helix pairing residue in the G helix, has been changed to Ala, Ser, Asp, and Leu. Stability to low-pH-induced unfolding has been measured for both native apoMb and the compact folding intermediate discovered by Griko et al. [Griko, Y. V., Privalov, P. L., Venyaminov, S. Y., & Kutyshenko, V. P. (1988) J. Mol. Biol. 202, 127-138]. As judged by its circular dichroism spectrum, this intermediate has a substantial helix content (about 35%). Whether or not this inferred helical structure is closely related to the myoglobin structure is not yet known. The mutational evidence shows that integrity of G.B helix pairing is important for the stability of apoMb as well as of myoglobin and that this helix pairing site is very sensitive to both steric and electrostatic disruption. Our results also suggest that G.B helix pairing does not stabilize the compact intermediate; hence, disrupting this site destabilizes the native protein relative to the compact intermediate. Such selective destabilization of the native state relative to equilibrium folding intermediates is not restricted to acid denaturation: urea denaturation of the Leu mutant appears to display at least one stable intermediate, while wild-type and the remaining mutant apoMbs undergo two-state urea unfolding transitions.
View details for Web of Science ID A1989U740100044
View details for PubMedID 2765493
The substitution Ala----Gly has been studied in a unique-sequence peptide (related in sequence to the C-peptide of ribonuclease A) to determine its effect on C-peptide helicity at different residue positions. There is a substantial decrease in helicity for Ala----Gly at residue position 4, 5, or 6 but only a small decrease in helicity for Ala----Gly at end residue 1 and no decrease at end residue 13. The change for Ala----Gly is similar at position 4, 5, or 6; the change is caused chiefly by the difference in s, the helix growth parameter in the Zimm-Bragg model for alpha-helix formation, between Ala and Gly. Thus, the helicity of C-peptide depends sensitively on s at interior positions. The small change in helicity found for Ala----Gly at either end position suggests that the end residues are largely excluded from the helix, with the result that helicity is relatively unaffected by replacement of an end residue. Another possibility is that some helix-stabilizing effect is exerted by Gly only at an end position. Exclusion of an end residue from the helix might be caused either by fraying of the helix ends or by helix termination at an interior residue, resulting from a helix stop signal such as the Glu-2- -Arg-10+ salt bridge or the Phe-8-His-12+ ring interaction.
View details for Web of Science ID A1989T673100025
View details for PubMedID 2719948
Interactions between the alpha-helix peptide dipoles and charged groups close to the ends of the helix were found to be an important determinant of alpha-helix stability in a previous study. The charge on the N-terminal residue of the C-peptide from ribonuclease A was varied chiefly by changing the alpha-NH2 blocking group, and the correlation of helix stability with N-terminal charge was demonstrated. An alternative explanation for some of those results is that the succinyl and acetyl blocking groups stabilize the helix by hydrogen bonding to an unsatisfied main-chain NH group. The helix dipole model is tested here with peptides that contain either a free alpha-NH3+ or alpha-COO- group, and no other charged groups that would titrate with similar pKa's. This model predicts that alpha-NH3+ and alpha-COO- groups are helix-destabilizing and that the destabilizing interactions are electrostatic in origin. The hydrogen bonding model predicts that alpha-NH3+ and alpha-COO- groups are not themselves helix-destabilizing, but that an acetyl or amide blocking group at the N- or C-terminus, respectively, stabilizes the helix by hydrogen bonding to an unsatisfied main-chain NH or CO group. The results are as follows: (1) Removal of the charge from alpha-NH3+ and alpha-COO- groups by pH titration stabilizes an alpha-helix. (2) The increase in helix stability on pH titration of these groups is close to the increase produced by adding an acetyl or amide blocking group.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1989U361900001
View details for PubMedID 2748569
The presence of an early intermediate on the folding pathway of ribonuclease A has been demonstrated by a study of the exchange reaction between the backbone amide protons in the folding protein and solvent protons using rapid mixing techniques. A structural analysis of the intermediate by two-dimensional 1H-NMR is consistent with the framework model of protein folding in which stable secondary structure first forms the framework necessary for the subsequent formation of the complete tertiary structure.
View details for Web of Science ID A1988Q532400046
View details for PubMedID 2845278
Four alanine-based peptides were designed, synthesized, and tested by circular dichroism for alpha-helix formation in H2O. Each peptide has three glutamic/lysine residue pairs, is 16 or 17 amino acids long, and has blocked alpha-NH2 and alpha-COOH groups. In one set of peptides ("i+4"), the glutamic and lysine residues are spaced 4 residues or 1 residue apart. In the other set ("i+3"), the spacing is 3 or 2 residues. Within each of these sets, a pair of peptides was made in which the positions of the glutamic and lysine residues are reversed [Glu, Lys (E,K) vs. Lys, Glu (K,E)] in order to assess the interaction of the charged side chains with the helix dipole. Since the amino acid compositions of these peptides differ at most by a single alanine residue, differences in helicity are caused chiefly by the spacing and positions of the charged residues. The basic aim of this study was to test for helix stabilization by (Glu-, Lys+) ion pairs or salt bridges (H-bonded ion pairs). The results are as follows. (i) All four peptides show significant helix formation, and the stability of the alpha-helix does not depend on peptide concentration in the range studied. The best helix-former is (i+4)E,K, which shows approximately 80% helicity in 0.01 M NaCl at pH 7 and 0 degree C. (ii) The two i+4 peptides show more helix formation than the i+3 peptides. pH titration gives no evidence for helix stabilization by i+3 ion pairs. (iii) Surprisingly, the i+4 peptides form more stable helices than the i+3 peptides at extremes of pH (pH 2 and pH 12) as well as at pH 7. These results may be explained by helix stabilization through Glu-...Lys+ salt bridges at pH 7 and singly charged H bonds at pH 2 (Glu0...Lys+) and pH 12 (Glu-...Lys0). The reason why these links stabilize the alpha-helix more effectively in the i+4 than in the i+3 peptides is not known. (iv) Reversal of the positions of glutamic and lysine residues usually affects helix stability in the manner expected for interaction of these charged groups with the helix dipole. (v) alpha-Helix formation in these alanine-based peptides is enthalpy-driven, as is helix formation by the C-peptide of ribonuclease A.
View details for Web of Science ID A1987L760100031
View details for PubMedID 3122208
Charged groups play a critical role in the stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A) in aqueous solution. One charged-group effect may arise from interactions between charged residues at either end of the helix and the helix dipole. We report here that studies of C-peptide analogues support the helix dipole model, and provide further evidence for the importance of electrostatic interactions not included in the Zimm-Bragg model for alpha-helix formation.
View details for Web of Science ID A1987G775600048
View details for PubMedID 3561498
Recent work has shown that, with synthetic analogues of C-peptide (residues 1-13 of ribonuclease A), the stability of the peptide helix in H2O depends strongly on the charge on the N-terminal residue. We have asked whether, in semisynthetic ribonuclease S reconstituted from S-protein plus an analogue of S-peptide (1-15), the stability of the peptide helix is correlated with the Tm of the reconstituted ribonuclease S. Six peptides have been made, which contain Glu9----Leu, a blocked alpha-COO- group (-CONH2), and either Gln11 or Glu11. The N-terminal residue has been varied; its charge varies from +2 (Lys) to -1 (succinyl-Ala). We have measured the stability of the peptide helix, the affinity of the peptide for S-protein (by C.D. titration), and the thermal stability of the reconstituted ribonuclease S. All six peptide analogues show strongly enhanced helix formation compared to either S-peptide (1-15) or (1-19), and the helix content increases as the charge on the N-terminal residue changes from +2 to -1. All six peptides show increased affinity for S-protein compared to S-peptide (1-19), and all six reconstituted ribonucleases S show an increase in Tm compared to the protein with S-peptide (1-19). The Tm increases as the charge on residue 1 changes from +2 to -1. The largest increment in Tm is 6 degrees. The results suggest that the stability of a protein can be increased by enhancing the stability of its secondary structure.
View details for PubMedID 3449849
A method for detecting structure in marginally stable forms of a protein is described. The principle is to measure amide proton exchange rates in the absence and presence of varying concentrations of a denaturant. Unfolding of structure by the denaturant is reflected by an acceleration of amide proton exchange rates, after correction for the effects of the denaturant on the intrinsic rate of exchange. This exchange-rate test for structure makes no assumptions about the rate of exchange in the unfolded state. The effects of 0-8 M urea and 0-6 M guanidinium chloride (GdmCl) on acid- and base-catalyzed exchange from model compounds have been calibrated. GdmCl does not appear to be well-suited for use in the exchange-rate test; model compound studies show that the effects of GdmCl on intrinsic exchange rates are complicated. In contrast, the effects of urea are a more uniform function of denaturant concentration. Urea increases acid-catalyzed, and decreases base-catalyzed, rates in model compounds. The exchange-rate test is used here to study structure formation in the S-protein (residues 21-124 of ribonuclease A). In conditions where an equilibrium folding intermediate of S-protein (I3) is known to be populated (pH 1.7, 0 degree C), the exchange-rate test for structure is positive. At higher temperatures (greater than 32 degrees C) I3 is unfolded, but circular dichroism data suggest that residual structure remains [Labhardt, A. M. (1982) J. Mol. Biol. 157, 357-371].(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1986A562900036
View details for PubMedID 3964684
The residues responsible for the pH-dependent stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A) have been identified by chemical synthesis of analogues and measurement of their helix-forming properties. Each of the residues ionizing between pH 2 and pH 8 has been replaced separately by an uncharged residue. Protonation of Glu-2- is responsible for the sharp decrease in helix stability between pH 5 and pH 2, and deprotonation of His-12+ causes a similar decrease between pH 5 and pH 8. Glu-9- is not needed for helix stability. The results cannot be explained by the Zimm-Bragg model and host-guest data for alpha-helix formation, which predict that the stability of the C-peptide helix should increase when Glu-2- is protonated or when His-12+ is deprotonated. Moreover, histidine+ is a strong helix-breaker in host-guest studies. In proteins, acidic and basic residues tend to occur at opposite ends of alpha-helices: acidic residues occur preferentially near the NH2-terminal end and basic residues near the COOH-terminal end. A possible explanation, based on a helix dipole model, has been given [Blagdon, D. E. & Goodman, M. (1975) Biopolymers 14, 241-245]. Our results are consistent with the helix dipole model and they support the suggestion that the distribution of charged residues in protein helices reflects the helix-stabilizing propensity of those residues. Because Glu-9 is not needed for helix stability, a possible Glu-9-...His-12+ salt bridge does not contribute significantly to helix stability. The role of a possible Glu-2-...Arg-10+ salt bridge has not yet been evaluated. A charged-group effect on alpha-helix stability in water has also been observed in a different peptide system [Ihara, S., Ooi, T. & Takahashi, S. (1982) Biopolymers 21, 131-145]: block copolymers containing (Ala)20 and (Glu)20 show partial helix formation at low temperatures, pH 7.5, where the glutamic acid residues are ionized. (Glu)20(Ala)20Phe forms a helix that is markedly more stable than (Ala)20(Glu)20Phe. The results are consistent with a helix dipole model.
View details for Web of Science ID A1985AGC7000033
View details for PubMedID 3857585
L.-N. Lin and J.F. Brandts recently proposed a simple model for the folding kinetics of ribonuclease A in which folding intermediates are not detectable. We have tested the basic assumption of the simple model for the major unfolded species, which is produced by a slow isomerization (the "X in equilibrium Y reaction" according to Lin and Brandts) after unfolding. The simple model assumes that in refolding the slow Y----X reaction must occur before any folding can take place. We have measured the Y----X reaction during folding. Tyrosine-detected folding occurs before the Y----X reaction; the difference in rate between the Y----X reaction and folding monitored by tyrosine absorbance becomes large when the stabilizing salt 0.56 M (NH4)2SO4 is added. The simple model predicts that the kinetic properties of the X in equilibrium Y reaction in unfolded ribonuclease are the same as those of tyrosine-detected folding. We find, however, that the kinetics of the X in equilibrium Y reaction in unfolded ribonuclease are independent of urea concentration, whereas the rate of tyrosine-detected folding decreases almost 100-fold between 0.3 and 5 M urea, as reported by Lin and Brandts. We point out that the kinetic properties of the X in equilibrium Y reaction in unfolded ribonuclease are characteristic of proline isomerization.
View details for Web of Science ID A1984TA45800002
View details for PubMedID 6466645
The isolated S-peptide (residues 1-20 of ribonuclease A) is known to show partial alpha-helix formation in aqueous solutions at low temperatures. We show here that the helix is limited to certain residues, including ones that are helical in the intact protein, and that a functional helix termination signal exists in the isolated peptide.
View details for Web of Science ID A1984SA08600033
View details for PubMedID 6694731
C-peptide, which contains the 13 NH2-terminal residues of RNase A, shows partial helix formation in water at low temperature (1 degree C, pH 5, 0.1 M NaCl), as judged by CD spectra; the helix is formed intramolecularly [Brown, J. E. & Klee, W. A. (1971) Biochemistry 10, 470-476]. We find that helix stability depends strongly on pH: both a protonated histidine (residue 12) and a deprotonated glutamate (residue 9 or 2 or both) are required for optimal stability. This information, together with model building, suggests that the salt bridge Glu-9- ... His-12+ stabilizes the helix. Formation of the helix is enthalpy driven [van't Hoff delta H, - 16Kcal/mol (1 cal = 4.18 J)] and the helix is not observed above 30 degrees C. Proton NMR data indicate that several side chains adopt specific conformations as the helix is formed. These results have two implications for the mechanism of protein folding. First, they indicate that short alpha-helices, stabilized by specific side-chain interactions within the helix, can be stable enough in water to function as folding intermediates. Second, they suggest that similar experiments with peptides of controlled amino acid sequence could be used to catalogue the intrahelix interactions that stabilize or destabilize alpha-helices in aqueous solution. These data might provide the code relating amino acid sequence to the locations of alpha-helices in proteins.
View details for Web of Science ID A1982NL32300010
View details for PubMedID 6283528
In the folding reaction of the slow-folding species (US) of ribonuclease A (RNase A), the slow isomerization of wrong proline isomers provides a suitable trap for kinetic folding intermediates at low temperatures (0--10 degrees C). Partly folded intermediates are known to accumulate before proline isomerization takes place, after which native RNase A is formed. We have been able to measure the protection from amide proton exchange which is provided by structure in the intermediates at different times along the folding pathway. Previous work has shown that, by labeling the amide protons of the unfolded protein before initiating refolding, an early folding intermediate can be detected. The new pulse-labeling method presented here can be used to label later folding intermediates. Our results indicate that, in conditions which strongly favor the native protein, intermediates are formed which provide protection against exchange. However, when folding is initiated in 2.5 M Gdn . HCl, 10 degrees C, pH 7.5, conditions in which folding goes to completion but there are no spectroscopically detectable intermediates, then no intermediates are detected by our method. Alternate minimal mechanisms for the folding of US are presented.
View details for Web of Science ID A1980KV90600027
View details for PubMedID 6258629
A transient intermediate (I3) observed previously in the unfolding of ribonuclease A has been studied by employing a sequential mixing instrument to populate selectively this species. This approach has made it possible both to determine the refolding behavior of this species and to characterize further the kinetics of its formation. (1) Formation of I3 represents the earliest detectable change in unfolding. (2) The loss of the 2'CMP binding site occurs in parallel with the exposure of the interior of the protein to solvent. (3) I3 is distinct from previously described intermediates in refolding. (4) Overall condensation of the protein to exclude solvent from the interior, as well as the formation of a substrate binding site, takes place in approximately 30 ms (pH 5.8, 47 degrees C), indicating that the formation of native structure can take place faster than had previously been supposed.
View details for Web of Science ID A1979GF46000009
View details for PubMedID 33695
In unfolded RNase A there is an interconversion between slow-folding and fast-folding forms (U(S) right harpoon over left harpoon U(F)) that is known to show properties characteristic of proline isomerization in model peptides. Here, we accept the evidence that U(S) molecules contain nonnative proline isomers and we ask about the isomerization of these proline residues during folding. The U(S) right harpoon over left harpoon U(F) reaction in unfolded RNase A is used both to provide data on the kinetics of proline isomerization in the unfolded protein and as the basis of an assay for measuring proline isomerization during folding.The tyrosine-detected folding kinetics at low temperatures have been compared to those of proline isomerization in unfolded RNase A. The comparison is based on the recent observation that the U(S) right harpoon over left harpoon U(F) kinetics are independent of guanidinium chloride concentration, so that they can be extrapolated to low guanidinium chloride concentrations, at which folding takes place. At 0 degrees C the tyrosine-detected folding reaction is 100-fold faster than the conversion of U(S) to U(F) in unfolded RNase A. Consequently, the folding reaction is not rate-limited by proline isomerization as it occurs in unfolded RNase A. An assay is given for proline isomerization during folding. The principle is that native RNase A yields U(F) on unfolding, whereas protein molecules that still contain nonnative proline isomers yield U(S). Unfolding takes place at 0 degrees C, at which proline isomerization is slow compared to unfolding. This assay yields two important results: (i) The kinetics of proline isomerization during folding are substantially faster than in unfolded RNase A-e.g., 40-fold at 0 degrees C. The mechanism of the rate enhancement is unknown. (ii) At low temperatures (0-10 degrees C), and also in the presence of (NH(4))(2)SO(4), the tyrosine-detected folding reaction occurs before proline isomerization and yields a folded intermediate I(N) that is able to bind the specific inhibitor 2'-CMP. The results demonstrate that a folding intermediate is spectrally detectable when folding occurs at low temperatures. They suggest that low temperatures provide suitable conditions for determining the kinetic pathway of folding by characterizing folding intermediates.
View details for Web of Science ID A1979JA38200032
View details for PubMedID 293712
Unfolded RNase A is known to contain an equilibrium mixture of two forms, a slow-folding form (U(1)) and a fast-folding form (U(2)). If U(1) is produced after unfolding by the slow cis-trans isomerization of proline residues about X-Pro imide bonds, then the formation of U(1) should be catalyzed by strong acids. Therefore, the rate of formation of U(1) has been measured at different HClO(4) concentrations. After rapid unfolding of the native protein in concentrated HClO(4) at 0 degrees , the slow formation of U(1) was measured by use of refolding assays. Catalysis of its formation was found at HClO(4) concentrations above 5 M. The uncatalyzed reaction follows apparent first-order kinetics but, in the acid-catalyzed range, two reactions are found. The faster reaction produces two-thirds of the slow-folding species and shows acid catalysis above 5 M HClO(4). Catalysis of the slower reaction begins at 8 M HClO(4). The faster reaction shows a 100-fold increase in rate at 10.6 M HClO(4) over the rate of the uncatalyzed reaction of 5 M. The activation enthalpy of the uncatalyzed reaction has been measured in two sets of unfolding conditions: DeltaH(double dagger) is 21.5 kcal/mol (1 kcal = 4.2 x 10(3) J) in 3.3 M HClO(4) and 21.0 kcal/mol in 5 M guanidine HCl, pH 2.5.Both acid catalysis of the formation of U(1) and its high activation enthalpy are consistent with the rate-limiting step being cis-trans isomerization either of X-Pro imide bonds or of peptide bond. The rate of the uncatalyzed reaction is in the range expected for proline isomerization and is 0.1% of that of peptide bond isomerization; thus, the simplest explanation for the formation of U(1) is proline isomerization. Earlier data, showing that the kinetic properties of the U(1) right arrow over left arrow U(2) reaction in refolding conditions differ from those of proline isomerization, can be explained if there is kinetic coupling between early steps in the folding of U(1) and its conversion to U(2).The existence of two acid-catalyzed reactions that are distinguished by the HClO(4) concentration at which catalysis begins suggests that at least two essential proline residues produce slow-folding species of RNase A by isomerization after unfolding. Because protonation of imide bonds is responsible for acid catalysis of proline isomerization, the slower reaction probably involves an imide bond with a low pK. It may be the bond connecting Lys-41 and Pro-42, because the positive charge on Lys-41 could make this bond more difficult to protonate.
View details for Web of Science ID A1978FU61400032
View details for PubMedID 283390