Effect of the N3 residue on the stability of the alpha-helix

N3 is the third position from the N terminus in the alpha-helix with helical backbone dihedral angles. All 20 amino acids have been placed in the N3 position of a synthetic helical peptide (CH(3)CO-[AAX AAAAKAAAAKAGY]-NH(2)) and the helix content measured by circular dichroism spectroscopy at 273 K. The dependence of peptide helicity on N3 residue identity has been used to determine a free energy scale by analysis with a modified Lifson-Roig helix coil theory that includes a parameter for the N3 energy (n3). The most stabilizing residues at N3 in rank order are Ala, Glu, Met/Ile, Leu, Lys, Ser, Gln, Thr, Tyr, Phe, Asp, His, and Trp. Free energies for the most destabilizing residues (Cys, Gly, Asn, Arg, and Pro) could not be fitted. The results correlate with N1, N2, and helix interior energies and not at all with N-cap preferences. This completes our work on studying the structural and energetic preferences of the amino acids for the N-terminal positions of the alpha-helix. These results can be used to rationally modify protein stability, help design helices, and improve prediction of helix location and stability.     

Effect of the N1 residue on the stability of the alpha-helix for all 20 amino acids

N1 is the first residue in an alpha-helix. We have measured the contribution of all 20 amino acids to the stability of a small helical peptide CH(3)CO-XAAAAQAAAAQAAGY-NH(2) at the N1 position. By substituting every residue into the N1 position, we were able to investigate the stabilizing role of each amino acid in an isolated context. The helix content of each of the 20 peptides was measured by circular dichroism (CD) spectroscopy. The data were analyzed by our modified Lifson-Roig helix-coil theory, which includes the n1 parameter, to find free energies for placing a residue into the N1 position. The rank order for free energies is Asp(-), Ala > Glu(-) > Glu(0) > Trp, Leu, Ser > Asp(0), Thr, Gln, Met, Ile > Val, Pro > Lys(+), Arg, His(0) > Cys, Gly > Phe > Asn, Tyr, His(+). N1 preferences are clearly distinct from preferences for the preceding N-cap and alpha-helix interior. pK(a) values were measured for Asp, Glu, and His, and protonation-free energies were calculated for Asp and Glu. The dissociation of the Asp proton is less favorable than that of Glu, and this reflects its involvement in a stronger stabilizing interaction at the N terminus. Proline is not energetically favored at the alpha-helix N terminus despite having a high propensity for this position in crystal structures. The data presented are of value both in rationalizing mutations at N1 alpha-helix sites in proteins and in predicting the helix contents of peptides.     

Effect of the N2 residue on the stability of the α-helix for all 20 amino acids

N2 is the second position in the alpha-helix. All 20 amino acids were placed in the N2 position of a synthetic helical peptide (CH(3)CO-[AXAAAAKAAAAKAAGY]-NH(2)) and the helix content was measured by circular dichroism spectroscopy at 273K. The dependence of peptide helicity on N2 residue identity has been used to determine a free-energy scale by analysis with a modified Lifson-Roig helix-coil theory that includes a parameter for the N2 energy (n2). The rank order of DeltaDeltaG((relative to Ala)) is Glu(-), Asp(-) > Ala > Glu(0), Leu, Val, Gln, Thr, Ile, Ser, Met, Asp(0), His(0), Arg, Cys, Lys, Phe > Asn, > Gly, His(+), Pro, Tyr. The results correlate very well with N2 propensities in proteins, moderately well with N1 and helix interior preferences, and not at all with N-cap preferences. The strongest energetic effects result from interactions with the helix dipole, which favors negative charges at the helix N terminus. Hydrogen bonds to side chains at N2, such as Gln, Ser, and Thr, are weak, despite occurring frequently in protein crystal structures, in contrast to the N-cap position. This is because N-cap hydrogen bonds are close to linear, whereas N2 hydrogen bonds have poor geometry. These results can be used to modify protein stability rationally, help design helices, and improve prediction of helix location and stability.     

Role of backbone hydration and salt-bridge formation in stability of alpha-helix in solution

We test molecular level hypotheses for the high thermal stability of alpha-helical conformations of alanine-based peptides by performing detailed atomistic simulations of a 20-amino-acid peptide with explicit treatment of water. To assess the contribution of large side chains to alpha-helix stability through backbone desolvation and salt-bridge formation, we simulate the alanine-rich peptide, Ac-YAEAAKAAEAAKAAEAAKAF-Nme, referred to as the EK peptide, that has three pairs of "i, i + 3" glutamic acid(-) and lysine(+) substitutions. Efficient configurational sampling of the EK peptide over a wide temperature range enabled by the replica exchange molecular dynamics technique allows characterization of the stability of alpha-helix with respect to heat-induced unfolding. We find that near ambient temperatures, the EK peptide predominately samples alpha-helical configurations with 80% fractional helicity at 300 K. The helix melts over a broad range of temperatures with melting temperature, T(m), equal to 350 K, that is significantly higher than the T(m) of a 21-residue polyalanine peptide, A(21). Salt-bridges between oppositely charged Glu(-) and Lys(+) side chains can, in principle, provide thermal stability to alpha-helical conformers. For the specific EK peptide sequence, we observe infrequent formation of Glu-Lys salt-bridges (with approximately 10-20% probability) and therefore we conclude that salt-bridge formation does not contribute significantly to the EK peptide's helical stability. However, lysine side chains are found to shield specific "i, i + 4" backbone hydrogen bonds from water, indicating that large side-chain substituents can play an important role in stabilizing alpha-helical configurations of short peptides in aqueous solution through mediation of water access to backbone hydrogen bonds. These observations have implications on molecular engineering of peptides and biomolecules in the design of their thermostable variants where the shielding mechanism can act in concert with other factors such as salt-bridge formation, thereby increasing thermal stability considerably.     

Reversal of negative charges on the surface of Escherichia coli thioredoxin: pockets versus protrusions

Recent studies of proteins with reversed charged residues have demonstrated that electrostatic interactions on the surface can contribute significantly to protein stability. We have used the approach of reversing negatively charged residues using Arg to evaluate the effect of the electrostatics context on the transition temperature (T(m)), the unfolding Gibbs free energy change (DeltaG), and the unfolding enthalpy change (DeltaH). We have reversed negatively charged residues at a pocket (Asp9) and protrusions (Asp10, Asp20, Glu85), all located in interconnecting segments between elements of secondary structure on the surface of Arg73Ala Escherichia coli thioredoxin. DSC measurements indicate that reversal of Asp in a pocket (Asp9Arg/Arg73Ala, DeltaT(m) = -7.3 degrees C) produces a larger effect in thermal stability than reversal at protrusions: Asp10Arg/Arg73Ala, DeltaT(m) = -3.1 degrees C, Asp20Arg/Arg73Ala, DeltaT(m) = 2.0 degrees C, Glu85Arg/Arg73Ala, DeltaT(m) = 3.9 degrees ). The 3D structure of thioredoxin indicates that Asp20 and Glu85 have no nearby charges within 8 A, while Asp9 does not only have Asp10 as sequential neighbor, but it also forms a 5-A long-range ion pair with the solvent-exposed Lys69. Further DSC measurements indicate that neutralization of the individual charges of the ion pair Asp9-Lys69 with nonpolar residues produces a significant decrease in stability in both cases: Asp9Ala/Arg73Ala, DeltaT(m) = -3.7 degrees C, Asp9Met/Arg73Ala, DeltaT(m) = -5.5 degrees C, Lys69Leu/Arg73Ala, DeltaT(m) = -5.1 degrees C. However, thermodynamic analysis shows that reversal or neutralization of Asp9 produces a 9-15% decrease in DeltaH, while both reversal of Asp at protrusions and neutralization of Lys69 produce negligible changes. These results correlate well with the NMR analysis, which demonstrates that only the substitution of Asp9 produces extensive conformational changes and these changes occur in the surroundings of Lys69. Our results led us to suggest that reversal of a negative charge at a pocket has a larger effect on stability than a similar reversal at a protrusion and that this difference arises largely from short-range interactions with polar groups within the pocket, rather than long-range interactions with solvent-exposed charged groups.     

Structural stability of wild type and mutated alpha-keratin fragments: molecular dynamics and free energy calculations

The objective of this study is to investigate the influence of point mutations on the structural stability of coiled coil fragments of the human hair intermediate filament by molecular dynamics simulations and free energy calculations. Mutations in the helix termination motif of human hair keratin gene hHb6 seem to be connected to the hereditary hair dystrophy Monilethrix. The most common mutations reported are Glu413Lys and Glu413Asp, located at the C-terminal end of the coiled coil 2B rod domain of the IF. According to our simulations, significant conformational changes of the side chains at the mutation and neighboring sites occur due to the Glu413Lys mutation. Furthermore, the differences in electrostatic interactions cause a large change in free energy during transformation of Glu413 to Lys calculated by the thermodynamic integration approach. It is speculated that the structural rearrangement necessary to adapt the interactions in the mutated coiled coil leads to changes in the IF assembly or its stability. The second mutation, Glu413Asp, only leads to a small value of the calculated free energy difference that is within the error limits of the simulations. Thus, it has to be concluded that this mutation does not affect the coiled coil stability.     

Surface electrostatic interactions contribute little of stability of barnase

Electrostatic interactions are believed to play an important role in stabilizing the native structure of proteins. We have quantified the contribution to stability of an interaction between two oppositely charged side-chains on the surface of barnase. Using site-directed mutagenesis, glutamate 28 and lysine 32 were introduced onto the solvent-accessible side of the second alpha-helix in barnase. These two residues are separated by one turn of the helix, and so are ideally situated for their opposite charges to interact. Double mutant cycle analysis reveals that the interaction between Glu28 and Lys32 contributes only approximately 0.2 kcal/mol to stability of the protein. All other interactions between exposed charged side-chains in barnase examined so far also contribute little to stability. We explain this low value by their location on the surface, rather than in the interior, of the protein.     

Structure, stability and folding of the alpha-helix

Pauling first described the alpha-helix nearly 50 years ago, yet new features of its structure continue to be discovered, using peptide model systems, site-directed mutagenesis, advances in theory, the expansion of the Protein Data Bank and new experimental techniques. Helical peptides in solution form a vast number of structures, including fully helical, fully coiled and partly helical. To interpret peptide results quantitatively it is essential to use a helix/coil model that includes the stabilities of all these conformations. Our models now include terms for helix interiors, capping, side-chain interactions, N-termini and 3(10)-helices. The first three amino acids in a helix (N1, N2 and N3) and the preceding N-cap are unique, as their amide NH groups do not participate in backbone hydrogen bonding. We surveyed their structures in proteins and measured their amino acid preferences. The results are predominantly rationalized by hydrogen bonding to the free NH groups. Stabilizing side-chain-side-chain energies, including hydrophobic interactions, hydrogen bonding and polar/non-polar interactions, were measured accurately in helical peptides. Helices in proteins show a preference for having approximately an integral number of turns so that their N- and C-caps lie on the same side. There are also strong periodic trends in the likelihood of terminating a helix with a Schellman or alpha L C-cap motif. The kinetics of alpha-helix folding have been studied with stopped-flow deep ultraviolet circular dichroism using synchrotron radiation as the light source; this gives a far superior signal-to-noise ratio than a conventional instrument. We find that poly(Glu), poly(Lys) and alanine-based peptides fold in milliseconds, with longer peptides showing a transient overshoot in helix content.     

The Glu 2- ... Arg 10+ side-chain interaction in the C-peptide helix of ribonuclease A

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.     

Conformational transitions and fibrillation mechanism of human calcitonin as studied by high-resolution solid-state 13C NMR

Conformational transitions of human calcitonin (hCT) during fibril formation in the acidic and neutral conditions were investigated by high-resolution solid-state 13C NMR spectroscopy. In aqueous acetic acid solution (pH 3.3), a local alpha-helical form is present around Gly10 whereas a random coil form is dominant as viewed from Phe22, Ala26, and Ala31 in the monomer form on the basis of the 13C chemical shifts. On the other hand, a local beta-sheet form as viewed from Gly10 and Phe22, and both beta-sheet and random coil as viewed from Ala26 and Ala31 were detected in the fibril at pH 3.3. The results indicate that conformational transitions from alpha-helix to beta-sheet, and from random coil to beta-sheet forms occurred in the central and C-terminus regions, respectively, during the fibril formation. The increased 13C resonance intensities of fibrils after a certain delay time suggests that the fibrillation can be explained by a two-step reaction mechanism in which the first step is a homogeneous association to form a nucleus, and the second step is an autocatalytic heterogeneous fibrillation. In contrast to the fibril at pH 3.3, the fibril at pH 7.5 formed a local beta-sheet conformation at the central region and exhibited a random coil at the C-terminus region. Not only a hydrophobic interaction among the amphiphilic alpha-helices, but also an electrostatic interaction between charged side chains can play an important role for the fibril formation at pH 7.5 and 3.3 acting as electrostatically favorable and unfavorable interactions, respectively. These results suggest that hCT fibrils are formed by stacking antiparallel beta-sheets at pH 7.5 and a mixture of antiparallel and parallel beta-sheets at pH 3.3.     

Analysis of the interaction between charged side chains and the alpha-helix dipole using designed thermostable mutants of phage T4 lysozyme

It was shown previously that the introduction of a negatively charged amino acid at the N-terminus of an alpha-helix could increase the thermostability of phage T4 lysozyme via an electrostatic interaction with the "helix dipole" [Nicholson, H., Becktel, W. J., & Matthews, B. W. (1988) Nature 336, 651-656]. The prior report focused on the two stabilizing substitutions Ser 38----Asp (S38D) and Asn 144----Asp (N144D). Two additional examples of stabilizing mutants, T109D and N116D, are presented here. Both show the pH-dependent increase in thermal stability expected for the interaction of an aspartic acid with an alpha-helix dipole. Control mutants were also constructed to further characterize the nature of the interaction with the alpha-helix dipole. High-resolution crystal structure analysis was used to determine the nature of the interaction of the substituted amino acids with the end of the alpha-helix in both the primary and the control mutants. Control mutant S38N has stability essentially the same as that of wild-type lysozyme but hydrogen bonding similar to that of the stabilizing mutant S38D. This confirms that it is the electrostatic interaction between Asp 38 and the helix dipole, rather than a change in hydrogen-bonding geometry, that gives enhanced stability. Structural and thermodynamic analysis of mutant T109N provide a similar control for the stabilizing replacement T109D. In the case of mutant N116D, there was concern that the enhanced stability might be due to a favorable salt-bridge interaction between the introduced aspartate and Arg 119, rather than an interaction with the alpha-helix dipole. The additivity of the stabilities of N116D and R119M seen in the double mutant N116D/R119M indicates that favorable interactions are largely independent of residue 119. As a further control, Asp 92, a presumed helix-stabilizing residue in wild-type lysozyme, was replaced with Asn. This decreased the stability of the protein in the manner expected for the loss of a favorable helix dipole interaction. In total, five mutations have been identified that increase the thermostability of T4 lysozyme and appear to do so by favorable interactions with alpha-helix dipoles. As measured by the pH dependence of stability, the strength of the electrostatic interaction between the charged groups studied here and the helix dipole ranges from 0.6 to 1.3 kcal/mol in 150 mM KCl. In the case of mutants S38D and N144H, NMR titration was used to measure the pKa's of Asp 38 and His 144 in the folded structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calculation of the electric potential in the active site cleft due to alpha-helix dipoles

A macroscopic dielectric model has been used to set up the electrostatic equation for the protein-solvent system. A numerical method of solution has been applied, enabling calculation of the electric potential outside a protein due to the charges within the protein. The glycolytic enzyme phosphoglycerate mutase, which is an α/β protein binding negatively charged substrates, has been studied. Modelling the helix dipoles with positive and negative charges shows that the α-helical structure could stabilize negatively charged substrates in the active site cleft of an enzyme with an energy of a few kT.     

Nature of amino acid side chain and alpha-helix stability.

In order to investigate the ability of neutral amino acids to support the α-helix conformation, the coil–helix transition of poly(L -lysine) and of lysine copolymers with these amino acids was studied in water/methanol using circular dichroism. The transtions were recorded at constant pH adding buffer to the methanol/water mixtures. With poly(L -lysine), experiments were performed at several constant pH's; the transition midpoint on the water (methanol) concentration scale was found to depend strongly upon pH; the helix stability region is shifted towards higher water concentrations, when the pH is increased. Copolymers of lysine and several neutral amino acids revealed the same effect in that increasing amounts of, for example, norleucine also shifted the transition midpoint to higher water concentrations. A series of copolymers containing L -lysine as the host and different hydrophobic amino acids were synthesized and the helix–coil transition in water/methanol was observed at constant pH. Different copolymers of equal composition showed significant differences with respect to the nature of the amino acid incorporated into polylysine. From these studies an α-helix-philic scale (in decreasing order): Leu, Nle, Ile, Ala, Phe, Val, Gly is deduced and discussed; the results obtained were compared with those of different procedures.     

Cumulative site-directed charge-change replacements in bacteriophage T4 lysozyme suggest that long-range electrostatic interactions contribute little to protein stability

Bacteriophage T4 lysozyme is a basic molecule with an isoelectric point above 9.0, and an excess of nine positive charges at neutral pH. It might be expected that it would be energetically costly to bring these out-of-balance charges from the extended, unfolded, form of the protein into the compact folded state. To determine the contribution of such long-range electrostatic interactions to the stability of the protein, five positively charged surface residues, Lys16, Arg119, Lys135, Lys147 and Arg154, were individually replaced with glutamic acid. Eight selected double, triple and quadruple mutants were also constructed so as to sequentially reduce the out-of-balance formal charge on the molecule from +9 to +1 units. Each of the five single variant proteins was crystallized and high-resolution X-ray analysis confirmed that each mutant structure was, in general, very similar to the wild-type. In the case of R154E, however, the Arg154 to Glu replacement caused a rearrangement in which Asp127 replaced Glu128 as the capping residue of a nearby alpha-helix. The thermal stabilities of all 13 variant proteins were found to be fairly similar, ranging from 0.5 kcal/mol more stable than wild-type to 1.7 kcal/mol less stable than wild-type. In the case of the five single charge-change variants, for which the structures were determined, the changes in stability can be rationalized in terms of changes in local interactions at the site of the replacement. There is no evidence that the reduction in the out-of-balance charge on the molecule increases the stability of the folded relative to the unfolded form, either at pH 2.8 or at pH 5.3. This indicates that long-range electrostatic interactions between the substituted amino acid residues and other charged groups on the surface of the molecule are weak or non-existent. Furthermore, the relative stabilities of the multiple charge replacement mutant proteins were found to be almost exactly equal to the sums of the relative stabilities of the constituent single mutant proteins. This also clearly indicates that the electrostatic interactions between the replaced charges are negligibly small. The activities of the charge-change mutant lysozymes, as measured by the rate of hydrolysis of cell wall suspensions, are essentially equal to that of the wild-type lysozyme, but on a lysoplate assay the mutant enzymes appear to have higher activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Position effect on apparent helical propensities in the C-peptide helix

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.     

Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges

Electrostatic interactions play a complex role in stabilizing proteins. Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper AB(SS). The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pK(a) induced by folding of the coiled coil structure. pK(a)(F) values of ten Glu and two His side-chains in folded AB(SS) and the corresponding pK(a)(U) values in unfolded peptides with partial sequences of AB(SS) were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (-5.6 kJ mol(-1)) and two are interacting with the positive alpha-helix dipoles and are thus stabilizing (+3.8 kJ mol(-1)) in charged form. The two His residues positioned in the C-terminal moiety of AB(SS) interact with the negative alpha-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (-2.6 kJ mol(-1)). Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (-1.1 kJ mol(-1)). The sum of the individual contributions of protonated Glu and His to the higher stability of AB(SS) at acidic pH (-5.4 kJ mol(-1)) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (-5.3 kJ mol(-1)). To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys-->norleucine, Arg-->norvaline). Both mutations destabilize the coiled coil conformation at neutral pH and increase the pK(a) of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing. Because removing charges by a double mutation cycle mainly discloses the immediate charge-charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability.     

Cooperative alpha-helix unfolding in a protein-DNA complex from hydrogen-deuterium exchange

We present experimental evidence for a cooperative unfolding transition of an alpha-helix in the lac repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen-deuterium (H-D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton HN to exchange. Close to this regime, we measured decay rates of individual backbone HN signals in D2O, and of their mutual HN-HN NOE by time-resolved two-dimensional (2D) NMR experiments. The data revealed correlated exchange at the center of the lac headpiece recognition helix, Val20-Val23, and suggested that the correlation breaks down at Val24, at the C terminus of the helix. A lower degree of correlation was observed for the exchange of Val9 and Ala10 at the center of helix 1, while no correlation was observed for Val38 and Glu39 at the center of helix 3. We conclude that HN exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the HN exchange in helix 3 is dominated by random local structure fluctuations.     

Further studies of the helix dipole model: effects of a free alpha-NH3+ or alpha-COO- group on helix stability

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)     

Positional dependence of the effects of negatively charged Glu side chains on the stability of two-stranded α-helical coiled-coils

The effects on protein stability of negatively charged Glu side chains at different positions along the length of the α-helix were investigated in the two-stranded α-helical coiled-coil. A native coiled-coil has been designed which consists of two identical 35 residue polypeptide chains with a heptad repeat Q_gV_aG_bA_cL_dQ_eK_f and a Cys residue at position 2 to allow the formation of an interchain 2-2′ disulphide bridge. This coiled-coil contains no intra- or interchain electrostatic interactions and served as a control for peptides in which Glu was substituted for Gln in the e or g heptad positions. The effect of the substitutions on stability was determined by urea denaturation at 20°C with the degree of unfolding monitored by circular dichroism spectroscopy. A Glu substituted for Gln near the N-terminus in each chain of the coiled-coil stabilizes the coiled-coil at pH 7, consistent with the charge–helix dipole interaction model. This stability increase is modulated by pH change and the addition of salt (KCl or guanidine hydrochloride), confirming the electrostatic nature of the effect. In contrast, Glu substitution in the middle of the helix destabilizes the coiled-coil because of the lower helical propensity and hydrophobicity of Glu compared with Gln at pH 7. Taking the intrinsic differences into account, the apparent charge–helix dipole interaction at the N-terminus is approximately 0.35 kcal/mol per Glu substitution. A Glu substitution at the C-terminus destabilizes the coiled-coil more than in the middle owing to the combined effects of intrinsic destabilization and unfavourable charge–helix dipole interaction with the negative pole of the helix dipole. The estimated destabilizing charge–helix dipole interaction of 0.08 kcal/mol is smaller than the stabilizing interaction at the N-terminus. The presence of a 2-2′disulphide bridge appears to have little influence on the magnitude of the charge–helix dipole interactions at either end of the coiled-coil. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Energetic contribution of solvent-exposed ion pairs to alpha-helix structure.

Understanding the role of amino acid side-chain interactions in forming secondary structure in proteins is useful for deciphering how proteins fold and for predicting folded structures of proteins from their sequence. Analysis of the secondary structure as a function of pH in two designed synthetic peptides with identical composition but different sequences, affords a quantitative estimate of the free energy contribution of a single ion pair to the stability of an isolated alpha-helix. One peptide contains repeated blocks of Glu4Lys4. The second has repeated blocks of Glu2Lys2. The former contains significant helical structure at neutral pH while the latter has none, based on ultraviolet light circular dichroism measurements and 1H nuclear magnetic resonance spectroscopy. The difference is attributed to formation of helix-stabilizing salt-bridges between Glu- and Lys+ spaced at i, i + 4 intervals in the former peptide. The free energy of formation of a single Glu(-)-Lys+ salt-bridge can be evaluated by using a statistical model of the helix-coil transition that explicitly includes salt-bridges: the result is -0.50(+/- 0.05) kcal/mol at 4 degrees C and neutral pH in 10 mM salt, in agreement with a value derived for a single salt-bridge in a helix on the surface of a globular protein.