Effects of Side Chains in Helix Nucleation Differ from Helix Propagation

Effects of side chains in helix nucleation differ from helix propagation

Stephen E. Miller, Andrew M. Watkins, Neville R. Kallenbach, and Paramjit S. Arora1

Department of Chemistry, New York University, New York, NY 10003

Edited* by S. Walter Englander, University of Pennsylvania, Philadelphia, PA, and approved March 25, 2014 (received for review December 11, 2013) Helix–coil transition theory connects observable properties of the of local sequence effects (14). The ability to differentiate indi- α-helix to an ensemble of microstates and provides a foundation vidual σ values from individual s values could provide a deeper for analyzing secondary structure formation in proteins. Classical understanding of the impact of individual side chains on helix models account for cooperative helix formation in terms of an formation than provided by current models (6, 12). energetically demanding nucleation event (described by the σ con- Here, we describe an approach that estimates the population stant) followed by a more facile propagation reaction, with corre- of N-terminal tripeptide sequences organized in an α-helix sponding s constants that are sequence dependent. Extensive nucleus as a function of individual guest residues. The key studies of folding and unfolding in model peptides have led to difference between this investigation and classical measures of the determination of the propagation constants for amino acids. propensity is that our approach assesses the ability of a given However, the role of individual side chains in helix nucleation has residue to favor or disfavor nucleation; literature propensities not been separately accessible, so the σ constant is treated as in- are largely derived by measuring how the stability of a preformed dependent of sequence. We describe here a synthetic model that helix responds to different residues in a peptide sequence. Nu- allows the assessment of the role of individual amino acids in helix cleation and propagation effects are intrinsically convoluted. The nucleation. Studies with this model lead to the surprising conclu- current approach offers a direct measure of helix nucleation and sion that widely accepted scales of helical propensity are not pre- allows nucleation effects to be interrogated independently of dictive of helix nucleation. Residues known to be helix stabilizers propagation. Although previous studies have implemented cor- or breakers in propagation have only a tenuous relationship to

rection factors to account for structural effects on nucleation in BIOPHYSICS AND residues that favor or disfavor helix nucleation. calculations of helix propensities, such as N-caps and C-caps COMPUTATIONAL BIOLOGY (15), no independent and general measure of nucleation has synthetic helices | helix propensity been available (6, 12, 16, 17). The results of our study are in fact surprising, as they suggest that steric effects in alkyl side chains, he α-helix is the most prevalent secondary structure in pro- such as those from β-branching, do not have a strong impact on Tteins and can form extremely rapidly. Helix formation is thus helix nucleation. This result stands in contrast to the observed crucial in early steps of protein folding, and a complete de- negative effects of β-branched residues on helix propagation (13, CHEMISTRY scription of the kinetics and thermodynamics of α-helix for- 18, 19). mation is fundamental for understanding protein folding (1). Theoretical models of the helix–coil transition consider helix Results and Discussion formation to proceed in two steps: initial nucleation of a helical Our approach to assess helix nucleation uses hydrogen bond sequence, denoted by the parameter σ in the Zimm–Bragg surrogate (HBS) α-helices in which the N-terminal main-chain notation (2), followed by more favorable helix propagation, hydrogen bond is replaced with a covalent bond (Fig. 1B) (20). In denoted by s parameters. This distinction identifies the ener- earlier reports, we described HBS helices in which the hydrogen getically unfavorable organization of three consecutive amino bond is replaced with a carbon–carbon bond (21). Characterization acid residues to form a helical nucleus as the slow step in helix – formation (2 4). Helix propagation, in contrast, refers to the Significance addition of the next hydrogen bond to a preformed helix (Fig. 1A). Zimm–Bragg or equivalent theories posit that formation Complete description of the kinetics and thermodynamics of of short peptide helices in water is unfavorable because the α-helix formation is fundamental to the understanding of large decrease in entropy required for nucleation is not adequately protein folding because α-helices are the most abundant class compensated by the enthalpic gain from forming a small number of secondary structures and are implicated in the earliest steps of hydrogen bonds. of the folding process. Kinetic models of protein folding sug- The ability of different peptide sequences to adopt helical gest that helix folding is rate-limited by formation of a nucleus conformations has been rigorously investigated, and the stability followed by rapid propagation. The influence of individual of α-helices can be estimated from widely used scales of helical residues on propagation has been evaluated in numerous propensity (5). These scales are important for our understanding model peptides and proteins. Here, we describe a synthetic of protein structure, folding, and function. The classical studies model that enables experimental assessment of the role of for determination of helix propensities have used peptides, – individual residues in helix nucleation. Our results suggest coiled-coils, and protein models for host guest studies in which that amino acids contribute differently to nucleation than a guest residue is systematically substituted at a site in a host to propagation. structure (6–12). Helix–coil transition theory then relates changes in the conformational stability to microscopic helix propensities or Author contributions: P.S.A. designed research; S.E.M. and A.M.W. performed research; s constants for individual residues. The results provide a quanti- S.E.M., A.M.W., N.R.K., and P.S.A. analyzed data; and S.E.M., A.M.W., N.R.K., and P.S.A. tative basis for evaluating the behavior of peptide and protein wrote the paper. helices; for example, the s constant for alanine is strongly helix The authors declare no conflict of interest. stabilizing, whereas those of valine and other β-branched residues *This Direct Submission article had a prearranged editor. are destabilizing (11, 13). However, even the most detailed 1To whom correspondence should be addressed. E-mail: [email protected]. investigations of the stability and dynamics of helix formation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. treat the energetic contribution of helix nucleation as independent 1073/pnas.1322833111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1322833111 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 faithfully mimic the conformation of canonical α-helices (21, 22). Importantly, HBS helices are able to inhibit intracellular protein–protein interactions mediated by α-helical domains, supporting the hypothesis that these compounds reproduce the structure and function of protein α-helices (23, 24). To analyze the effect of different residues on α-helix formation, we prepared an HBS analog with a disulfide linkage whose rates of formation could be monitored under aqueous conditions (25). We con- jectured that the rates of formation of this linkage would provide a unique probe for examining biophysical parameters related to helix formation. Because the disulfide–HBS (dsHBS) linkage mimics the intramolecular hydrogen bond in α-helices, we hy- pothesized that the bisthiol (bt) to disulfide (ds) oxidation would provide a direct measure of helix nucleation (Fig. 1C). Rates of helix formation for model peptides have been measured by ul- trafast spectroscopies and fall in the nanosecond range (14, 26– 29). The rates of disulfide formation are much slower (on the order of minutes) under our reaction conditions (30). The dra- matically slower timescale for disulfide formation suggests that sequence-dependent differences in the rate of disulfide formation reflect preequilibrium populations: residues with higher nucleating propensity favor disulfide formation. Disulfide-linked HBS helices can be derived from oxidation of the parent bisthiol peptides allowing a facile approach to control the conformation of the peptide (31–34). We based our initial peptide design on a short segment from the p53 activation domain: XQEG*FSDLWKLLS (1), where X and G* represent the dsHBS-constrained residues (35). The p53 sequence was chosen because it has relatively few side-chain contacts such as ionic bridges, which may bias the results. We have previously charac- terized a number of p53 HBS analogs by CD and NMR spectroscopies allowing us to predict potential aggregation issues that can affect stabilized constructs (36–38). The bisthiol p53 peptide is weakly helical in aqueous buffers at 295 K. CD spectroscopy (Fig. 2A) shows that the p53 dsHBS 1 is highly helical compared with the parent bt-peptide (25). We selected A, G, D, I, K, L, P, and V as the guest residues for this investigation. This selection includes representative aliphatic straight chain and β-branched residues along with positively and negatively charged side chains. After initial experiments, peptide 1 was modified by replacing the native glutamic acid and phenylalanine residues with lysine and alanine, respectively, to obtain 2Λ:XΛKG*ASDLWKLLS. The new sequence was designed to avoid potential side-chain interactions between the guest residues (Λ) and the corresponding i + 3 position; lysine was incorporated to increase water solubility of the host. The second position was chosen for the introduction of guests because the conformation of this residue is implicated in helix induction near the N terminus (39, 40). The helical conformation of ds-2A: XAKG*ASDLWKLLS was assessed using a combination of 1D NMR, total correlation spectroscopy, and rotating-frame nuclear Overhauser effect correlation spectroscopy (ROESY) studies in aqueous solutions (SI Text, Table S1, and Fig. S1). The ROESY spectra revealed α nuclear Overhauser effect (NOE) cross-peaks indicative of a Fig. 1. Preorganization of three residues into an -turn conformation is the i i + energy-demanding step in helix formation. (A) Models of the helix–coil helical structure, including sequential NH-NH ( and 1) and transition consider helix formation to proceed in two steps consisting of several longer range NOEs (between i and i + 3/i + 4) (SI Text, nucleation and propagation steps. (B) In hydrogen bond surrogate (HBS) Table S2). Importantly, all backbone ϕ dihedral angles, calcu- α + 3 -helices, the nucleus is organized by replacement of a main chain i to i 4 lated from JNHCHα coupling constants, are in the expected range hydrogen bond with a covalent bond. The dsHBS helices feature a reversible for helical conformations (SI Text, Table S1) (41, 42). The cou- disulfide linkage. (C) Possible intermediates for the conversion of bisthiol I to pling constants observed for the residues within the macrocycle α dsHBS IV include bisthiol II in -turn conformation and disulfide III as the do not differ from the rest of the chain, indicating that the constrained helix nucleus. Rates of conversion of bisthiol to disulfide were disulfide constraint is faithfully inducing an α-helical conforma- measured as a function of variable residue Λ. tion. The solution structure of ds-2A was determined from 48 ROESY cross-peaks and 11 calculated ϕ angles using Monte Carlo conformational search in Macromodel 2014. An overlay of these synthetic helices by CD and 2D-NMR spectroscopy, as of the 20 lowest energy structures for ds-2A isshowninFig.2B. well as single crystal X-ray diffraction analysis, reveals that they Notably, the disulfide-constrained macrocycle does not perturb the

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Fig. 2. Conformational analysis and synthesis of unconstrained peptides. (A) CD spectroscopy suggests that the disulfide-linked (ds) HBS peptide is highly

helical compared with the parent bisthiol (bt). The CD studies were performed with 50 μM peptides in 1 mM PBS. (B) NMR-derived structures of ds-2A. Views CHEMISTRY of 20 lowest energy structures are shown with carbon, nitrogen, and oxygen atoms in gray, blue, and red, respectively. The disulfide linkage is shown in yellow color. (C) Schematic for the oxidation reaction. The conversion of bt-2Λ → ds-2Λ was affected under mild oxidative conditions; the unreacted bisthiol was quenched with maleimide 3.

Nterminusoftheα-helix. Overall, together with CD spectroscopy, analysis of HPLC peak areas (Fig. 3A and Fig. S3). The initial the NMR studies confirm stabilization of a major α-helical rates of disulfide formation are plotted in Fig. 3B, and the tab- conformation in dsHBS-constrained peptides. ulated data are presented in Table 1. The bisthiol and HBS peptides were synthesized as shown in We find that the nucleation propensity of alanine is stronger SI Text, Fig. S2. We used mild oxidation conditions consisting of than that of glycine, whereas the disulfide bond formation with 2% (vol/vol) DMSO to oxidize the bisthiols (Fig. 2C) (25, 30). To proline as guest is too slow to measure accurately. These results separate the closely related bisthiol and dsHBS peptides on are consistent with known relationships: glycine is a highly flex- HPLC and to quench the thiol groups, we treated the reaction ible residue, whereas proline aids helicity only as an N-cap res- mixture with a maleimide derivative (43). Adequate separation idue and not at other positions in the helix. These results also of the bisthiol and disulfide peptides on HPLC allowed us to indicate that conformation of the macrocycle influences rates of quantify the rates of disulfide formation for each sequence from the disulfide formation. If the rates of the bond formation were

Table 1. Comparison of dsHBS rate of formation to literature helix propensity values Rate of bt → ds conversion, ΔΔG from propensity, Peptide† μM/min‡ Rates relative to 2G kcal/mol§

XAKG*ASDLWKLLS (2A) 0.20 ± 0.02 2.86 −1.88 -–D––––––––––––––– (2D) 0.06 ± 0.01 0.86 −1.00 -–G––––––––––––––– (2G) 0.07 ± 0.005 1.00 0.00 -–I–––––––––––––––– (2I) 0.15 ± 0.01 2.14 −1.18 -–K––––––––––––––– (2K) 0.08 ± 0.01 1.14 −1.52 -–L––––––––––––––– (2L) 0.19 ± 0.01 2.71 −1.60 -–P––––––––––––––– (2P) –– –– >5.00 -–V––––––––––––––– (2V) 0.20 ± 0.01 2.86 −0.83

† X and G* represent the dsHBS constraint. ‡ Calculated from HPLC analysis. §From ref. 19.

Miller et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 independent of the macrocycle conformation, substitution nucleation. Surprisingly, our investigations reveal that β-branched of a single glycine in place of an alanine would not cause residues are not detrimental to α-helix nucleation. Indeed, the a significant effect. It is likely that the peptide chain beyond rate of the disulfide formation with valine is nearly the same as the putative macrocycle is influencing the preequilibrium that of alanine and leucine, whereas isoleucine leads to a slight conformation but because the bisthiol peptide is largely non– decrease. Charged residues, lysine and aspartate, slow down the α-helical, we expect this effect to be subtle and equivalent for reaction rate to the level of glycine. Because aspartic acid is a every mutant. known helix breaker whereas lysine is considered to be a helix Leucine and alanine sequences are similar in their disulfide stabilizing residue (44, 45), these results suggest that charged formation rates, as might be expected from the literature pro- residues potentially participate in long-range interactions. Al- pensity values (Table 1) (6). These results provide useful metrics though the sequences of this study were designed to minimize to gauge the potential of our synthetic model as a probe of helix context dependence, backbone interactions cannot be completely ruled out. CD spectroscopy shows that five of the eight dsHBS sequences have very similar helical content; substitution of glycine, proline, and aspartic acid lowers the helicity (Fig. 3C). We conjecture that helical stability of the constrained sequence is not a major determinant of disulfide rate formation. Comparison of the lysine and aspartic acid sequences provides support for this hypothesis, because these sequences display similar rates of disulfide formation, whereas the constrained peptides show different helical stabilities. Charged residues at the end of helices can positively or negatively influence helix stability based on their interactions with the helix macrodipole (46). However, such macrodipolar interactions should not influence the process of helix nucleation because the helix is not yet organized. To obtain theoretical support for our experimental observations, we calculated activation barriers to formation of an i to i + 4 hydrogen bond in peptide sequences using metadynamics simulations (47, 48). Helix–coil transitions have been previously investigated using molecular-dynamics simulations to analyze helix propensity and nucleation timescales, but, to our knowledge, the effect of point mutations on helix nucleation has not been ex- plored systematically (28, 39, 49). We determined the impact of different residues on the formation of an α-turn in an acetylated and methylamidated model peptide sequence, Ac-AΛA-NHMe. We used Schrodinger’s 2012 distribution of Desmond 3.1 (50) to conduct 50-ns metadynamics simulation on the tripeptide using the Amber forcefield ff12SB and associated monovalent ion parameters (51, 52). Two-dimensional free-energy surfaces were determined and activation energies were identified by in- spection (Fig. 4). The activation energies stabilize as simulation length increases, suggesting that the simulation converged at 15 ns (Fig. S4). We also performed the simulations in triplicate and measured the average rmsd between the resulting free-energy surfaces (Fig. S4). Analysis reveals that all guest (Λ) residues, except proline, sample dihedral angles corresponding to left- and right- handed α-helix, β-strand, and polyproline II (PPII) regions of the Ramachandran plot (Fig. 4A). The plot of the proline containing tripeptide features only the α and PPII minima (Fig. 4B). The [ϕ,ψ] plots for the other 18 guest residues in Ac-AΛA-NHMe are shown in Fig. S5. The weighted fraction of α, β, and PPII populations for different guest residues is plotted in Fig. 4C.Tocalculate the barrier for helix formation, we compared the height of the shortest peak between the lowest energy basin corresponding to the PPII or β dihedral angles to the α-helical dihedral for each residue (Fig. 4D). The PPII dihedral angles were found to be the lowest energy conformation for most guest residues in our calculations. The results of these calculations correlate with the experimental data and suggest that the barrier to preorganization of the α-helical conformation does not follow propensity scales. Fig. 3. Analysis of bisthiol to disulfide conversion. (A) Rates of the bt-to-ds Although the activation energies reflect starting populations and conversion were analyzed by HPLC, with tryptophan as an internal control. energy profiles of the α and β or PPII conformations, they pro- Λ = Representative HPLC results for peptide 2 with alanine ( A) as the guest vide a gauge for estimating the residue-dependent difficulty of residue are shown. (B) Plots of bt-to-ds conversion for different guest resi- α ΔG‡ dues. (C) CD spectra of sequences with different guest residues illustrate that adopting an -helical conformation. The calculated values the helical content of most sequences, with the exception of sequences with identify guest residues with fast and slow folding rates but Λ = G, P, and D, is identical. The CD studies were performed with 50 μM suggest that rates of several others are not readily distin- peptides in 5 mM KF, pH 7.3. guishable. Overall, the calculations support the experimental

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1322833111 Miller et al. Downloaded by guest on October 1, 2021 Fig. 4. Results of metadynamics simulation to evaluate barrier to the formation of an i to i + 4 hydrogen bond in a model peptide (AcAΛA-NHMe) as a function of different guest residues. (A and B) Two-dimensional free-energy surfaces for Λ = alanine and proline are shown with the others included in SI

Text. The Ramachandran plots of all residues show low energy basins for the α, β, and polyproline II (PPII) dihedral space with the exception of proline, where BIOPHYSICS AND α α β the PPII and spaces dominate. (C) Weighted populations of all low-energy basins corresponding to the , , and PPII dihedral angles. (D) The activation COMPUTATIONAL BIOLOGY energy barrier between the lowest energy region corresponding to the PPII space and α-helical dihedral angles.

observations that helical propensity scales are not applicable equipped with a reversed-phase C-18 column, and buffers containing 0.1% to helix nucleation. TFA in acetonitrile and 0.1% TFA in water. Purified peptides were analyzed by liquid chromatography (LC)/MS on an Agilent 1100 Series LCMSD system.

Conclusions High-resolution MS data were obtained using an Agilent 6224 Accurate- CHEMISTRY Mass TOF LC/MS. HPLC analysis for the conversion of bt to ds peptides was We have developed a synthetic model to evaluate propensities α performed using an Agilent 1200 series HPLC system with a Thermo Hypersil for helix nucleation. Although the organization of an -turn C-8 column (50 × 2mm,3μm; 5–65% acetonitrile in water over 10 min). CD leading to an intramolecular hydrogen bond between the C=O spectra for dsHBS helices were recorded on an AVIV 202SF CD spectrometer of the ith residue and the amide NH of the i + 4th residue equipped with a temperature controller using a 1-mm length cell and a scan is considered the slow step in helix formation, there are no speed of 5 nm/min. Samples were run in 5 mM KF buffer (pH 7.3) or 0.1× PBS reported experimental attempts to segregate effects of individual buffer (pH 7.3) at a concentration of 50 μM ds-2Λ (concentration determined e = −1· −1 side chains on helix nucleation. A large number of studies have by Trp residue absorbance, 5,560 cm M at 280 nm). The spectra were averaged over 10 scans with the baseline subtracted from analogous con- focused on the propensity of amino acid residues to propagate ditions to those of the samples. NMR experiments were performed using a preformed helix in peptide and protein models. To experi- a Bruker Avance 600-MHz spectrometer. IR analysis was performed using mentally interrogate the nucleation step, we replaced the intra- a Thermo Nicolet 6700 FT-IR spectrometer, equipped with Smart ITR. Solids molecular hydrogen bond at the N-terminus of a short α-helix by were dissolved in methanol and dried on smart sensor. a disulfide linkage whose rates of formation from the analogous bisthiol could be measured as a function of individual residues in Representative Procedure for the Oxidation of bt-2Λ → ds-2Λ. Disulfide pep- the turn. We also performed metadynamics simulations on tri- tides are prone to oxidation and were stored under inert atmosphere as peptide sequences to estimate the barrier to formation of a hy- lyophilized powders. Concentrations of purified bt-2Λ peptides were de- drogen bond between the ith and the i + 4th residues. termined under acidic conditions (0.1% aqueous TFA) to minimize oxidation. Lyophilized bisthiol peptides (200 nmol) were dissolved in 40 μL of DMSO in The thrust of these experimental and computational studies a centrifuge tube, and oxidation was initiated by addition of 2 mL of oxi- reveals for the first time (to our knowledge) that helix nucleation dation buffer (800 mM ammonium bicarbonate, 840 mM acetic acid, pH 6.0). differs from propagation in an important aspect: although fold- The oxidation buffer was supplemented with 50 μM tryptophan as an ing of a chain to create a nucleus is sensitive to the effects of internal control for HPLC studies. Fifty-microliter aliquots were removed a rigid template (as observed in crystal packing and growth), the from the reaction mixture after set intervals (T = 0, 1, 2, 5, 10, 20, and 30 μ process of nucleation minimizes what must be relatively minor min), quenched with 25 L of 1.4 mM maleimide 3 in H2O, and analyzed enthalpic and entropic penalties due to side-chain packing. The by HPLC. large entropic barrier that must be overcome to organize three Metadynamics Simulations. Acetylated and methylamidated peptide struc- consecutive residues predominatesinnucleationinmarked tures were initialized with tleap (AmberTools12) using the ff12SB force field contrast to the side-chain steric constraints that impact helix and monovalent ion parameters from Joung and Cheatham (51, 52). Struc- propagation (18). tures were neutralized by adding chloride or sodium ions and surrounded by a truncated octahedron of TIP3P water with a buffer distance of 8.0 Å. The Methods resulting topology and input coordinates were converted to Desmond cms General. Commercial-grade reagents and solvents were used without further format for simulations with Desmond 3.1. purification, except as indicated. Peptides were purified using a Beckman The ϕ and ψ dihedral angles of the guest residue were chosen as the Coulter HPLC equipped with a System Gold 168 Diode array detector, reaction coordinates for metadynamics column variables. The standard

Miller et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 Desmond relaxation protocol was conducted on each tripeptide: 2,000 steps minimum energy conformation was identified and the activation energy minimization with 10 steps steepest descent with 50 kcal/mol restraints on was calculated. peptides, 2,000 steps minimization with 10 steps steepest descent without restraints, 12 ps of Berendsen NVT simulation at 10 K, and finally 24 ps of ACKNOWLEDGMENTS. We thank David Rooklin and Yingkai Zhang for their Berendsen NVT equilibration at 300 K. A 50-ns metadynamics simulation was advice on metadynamics calculations, Stephen Joy for assistance with conducted on each peptide, depositing 5°-wide, 0.01-kcal-high Gaussians the structure calculations, and the National Institutes of Health (Grant every 12 fs. The resulting 2D free-energy surfaces were analyzed manually. R01GM073943) for financial support. A.M.W. is supported by a Margaret The transition state connecting the α-helical conformation to the nonhelical Strauss Kramer fellowship.

1. Dill KA, MacCallum JL (2012) The protein-folding problem, 50 years on. Science 28. De Sancho D, Best RB (2011) What is the time scale for α-helix nucleation? J Am Chem 338(6110):1042–1046. Soc 133(17):6809–6816. 2. Zimm BH, Bragg JK (1959) Theory of the phase transition between helix and random 29. Huang C-Y, Klemke JW, Getahun Z, DeGrado WF, Gai F (2001) Temperature- coil in polypeptide chains. J Chem Phys 31(2):526–535. dependent helix-coil transition of an alanine based peptide. J Am Chem Soc 123(38): 3. Qian H, Schellman JA (1992) Helix-coil theories: A comparative study for finite length 9235–9238. – preferences. J Phys Chem 96(10):3987 3994. 30. Tam JP, Wu CR, Liu W, Zhang JW (1991) Disulfide bond formation in peptides by 4. Lifson S, Roig A (1961) On the theory of helix-coil transitions in polypeptides. J Chem dimethyl sulfoxide. Scope and applications. J Am Chem Soc 113(17):6657–6662. – Phys 34(6):1963 1974. 31. Haney CM, Loch MT, Horne WS (2011) Promoting peptide α-helix formation with 5. Munoz V, Serrano L (1997) Development of the multiple sequence approximation dynamic covalent oxime side-chain cross-links. Chem Commun 47(39):10915–10917. within the AGADIR model of alpha-helix formation: Comparison with Zimm-Bragg 32. Bredenbeck J, Helbing J, Kumita JR, Woolley GA, Hamm P (2005) α-Helix formation in and Lifson-Roig formalisms. Biopolymers 41(5):495–509. a photoswitchable peptide tracked from picoseconds to microseconds by time-re- 6. Rohl CA, Chakrabartty A, Baldwin RL (1996) Helix propagation and N-cap propensities solved IR spectroscopy. Proc Natl Acad Sci USA 102(7):2379–2384. of the amino acids measured in alanine-based peptides in 40 volume percent tri- 33. Ihalainen JA, et al. (2008) α-Helix folding in the presence of structural constraints. Proc fluoroethanol. Protein Sci 5(12):2623–2637. Natl Acad Sci USA 105(28):9588–9593. 7. Blaber M, et al. (1994) Determination of alpha-helix propensity within the context of 34. Li M, Yamato K, Ferguson JS, Gong B (2006) Sequence-specific association in aqueous a folded protein. Sites 44 and 131 in bacteriophage T4 lysozyme. J Mol Biol 235(2): 600–624. media by integrating hydrogen bonding and dynamic covalent interactions. JAm – 8. Wojcik J, Altmann KH, Scheraga HA (1990) Helix-coil stability constants for the nat- Chem Soc 128(39):12628 12629. urally occurring amino acids in water. XXIV. Half-cystine parameters from random 35. Kussie PH, et al. (1996) Structure of the MDM2 oncoprotein bound to the p53 tumor poly(hydroxybutylglutamine-CO-S-methylthio-L-cysteine). Biopolymers 30(1-2):121–134. suppressor transactivation domain. Science 274(5289):948–953. 9. Padmanabhan S, Marqusee S, Ridgeway T, Laue TM, Baldwin RL (1990) Relative helix- 36. Patgiri A, Joy ST, Arora PS (2012) Nucleation effects in peptide foldamers. J Am Chem forming tendencies of nonpolar amino acids. Nature 344(6263):268–270. Soc 134(28):11495–11502. 10. O’Neil KT, DeGrado WF (1990) A thermodynamic scale for the helix-forming ten- 37. Mahon AB, Arora PS (2012) Design, synthesis and protein-targeting properties of dencies of the commonly occurring amino acids. Science 250(4981):646–651. thioether-linked hydrogen bond surrogate helices. Chem Commun (Camb) 48(10): 11. Lyu PC, Liff MI, Marky LA, Kallenbach NR (1990) Side chain contributions to the sta- 1416–1418. bility of alpha-helical structure in peptides. Science 250(4981):669–673. 38. Henchey LK, Porter JR, Ghosh I, Arora PS (2010) High specificity in protein recognition 12. Richardson JM, Lopez MM, Makhatadze GI (2005) Enthalpy of helix-coil transition: by hydrogen-bond-surrogate α-helices: Selective inhibition of the p53/MDM2 com- Missing link in rationalizing the thermodynamics of helix-forming propensities of the plex. ChemBioChem 11(15):2104–2107. – amino acid residues. Proc Natl Acad Sci USA 102(5):1413 1418. 39. Young WS, Brooks CL, 3rd (1996) A microscopic view of helix propagation: N and C- 13. Lyu PC, Sherman JC, Chen A, Kallenbach NR (1991) Alpha-helix stabilization by natural terminal helix growth in alanine helices. J Mol Biol 259(3):560–572. and unnatural amino acids with alkyl side chains. Proc Natl Acad Sci USA 88(12): 40. Cochran DAE, Doig AJ (2001) Effect of the N2 residue on the stability of the α-helix for 5317–5320. all 20 amino acids. Protein Sci 10(7):1305–1311. 14. Neumaier S, Reiner A, Büttner M, Fierz B, Kiefhaber T (2013) Testing the diffusing 41. Wang Y, Nip AM, Wishart DS (1997) A simple method to quantitatively measure boundary model for the helix-coil transition in peptides. Proc Natl Acad Sci USA polypeptide JHNH α coupling constants from TOCSY or NOESY spectra. J Biomol NMR 110(32):12905–12910. 10(4):373–382. 15. Aurora R, Rose GD (1998) Helix capping. Protein Sci 7(1):21–38. 42. Pardi A, Billeter M, Wüthrich K (1984) Calibration of the angular dependence of the 16. Lopez MM, Chin D-H, Baldwin RL, Makhatadze GI (2002) The enthalpy of the alanine amide proton-C alpha proton coupling constants, 3JHN alpha, in a globular protein. peptide helix measured by isothermal titration calorimetry using metal-binding to induce helix formation. Proc Natl Acad Sci USA 99(3):1298–1302. Use of 3JHN alpha for identification of helical secondary structure. J Mol Biol 180(3): – 17. Sun JK, Penel S, Doig AJ (2000) Determination of α-helix N1 energies after addition of 741 751. N1, N2, and N3 preferences to helix/coil theory. Protein Sci 9(4):750–754. 43. Brantley RK, Haeffner-Gormley L, Wetlaufer DB (1984) Preparation of a positively 18. Creamer TP, Rose GD (1992) Side-chain entropy opposes alpha-helix formation but charged maleimide and its application to the high-performance liquid chromato- rationalizes experimentally determined helix-forming propensities. Proc Natl Acad Sci graphic separation of the tryptic peptides of lysozyme. J Chromatogr A 295(1): USA 89(13):5937–5941. 220–225. 19. Chakrabartty A, Kortemme T, Baldwin RL (1994) Helix propensities of the amino acids 44. Huyghues-Despointes BM, Scholtz JM, Baldwin RL (1993) Effect of a single aspartate measured in alanine-based peptides without helix-stabilizing side-chain interactions. on helix stability at different positions in a neutral alanine-based peptide. Protein Sci Protein Sci 3(5):843–852. 2(10):1604–1611. 20. Patgiri A, Jochim AL, Arora PS (2008) A hydrogen bond surrogate approach for sta- 45. Yang JX, Zhao K, Gong YX, Vologodskii A, Kallenbach NR (1998) Alpha-helix nucle- bilization of short peptide sequences in alpha-helical conformation. Acc Chem Res ation constant in copolypeptides of alanine and ornithine or lysine. J Am Chem Soc – 41(10):1289 1300. 120(41):10646–10652. 21. Wang D, Chen K, Kulp JL, III, Arora PS (2006) Evaluation of biologically relevant short 46. Shoemaker KR, Kim PS, York EJ, Stewart JM, Baldwin RL (1987) Tests of the helix alpha-helices stabilized by a main-chain hydrogen-bond surrogate. J Am Chem Soc dipole model for stabilization of alpha-helices. Nature 326(6113):563–567. 128(28):9248–9256. 47. Laio A, Gervasio FL (2008) Metadynamics: A method to simulate rare events and re- 22. Liu J, Wang D, Zheng Q, Lu M, Arora PS (2008) Atomic structure of a short alpha-helix construct the free energy in biophysics, chemistry and material science. Rep Prog Phys stabilized by a main chain hydrogen-bond surrogate. J Am Chem Soc 130(13): 71(12):126601. 4334–4337. 48. Ensing B, De Vivo M, Liu Z, Moore P, Klein ML (2005) Metadynamics as a tool for 23. Kushal S, et al. (2013) Protein domain mimetics as in vivo modulators of hypoxia-in- exploring free energy landscapes of chemical reactions. Acc Chem Res 39(2):73–81. ducible factor signaling. Proc Natl Acad Sci USA 110(39):15602–15607. 49. Daggett V, Levitt M (1992) Molecular dynamics simulations of helix denaturation. 24. Patgiri A, Yadav KK, Arora PS, Bar-Sagi D (2011) An orthosteric inhibitor of the Ras- – Sos interaction. Nat Chem Biol 7(9):585–587. J Mol Biol 223(4):1121 1138. 25. Miller SE, Kallenbach NR, Arora PS (2012) Reversible α-helix formation controlled by 50. Bowers KJ, et al. (2006) Scalable algorithms for molecular dynamics simulations on a hydrogen bond surrogate. Tetrahedron 68(23):4434–4437. commodity clusters. Proceedings of the 2006 ACM/IEEE Conference on Super- 26. Serrano AL, Tucker MJ, Gai F (2011) Direct assessment of the α-helix nucleation time. computing (ACM,Tampa,FL),p84. J Phys Chem B 115(22):7472–7478. 51. Case DA (2012) AMBER 12 (Univ of California, San Francisco). 27. Lin MM, Mohammed OF, Jas GS, Zewail AH (2011) Speed limit of protein folding 52. Joung IS, Cheatham TE, 3rd (2008) Determination of alkali and halide monovalent ion evidenced in secondary structure dynamics. Proc Natl Acad Sci USA 108(40): parameters for use in explicitly solvated biomolecular simulations. J Phys Chem B 16622–16627. 112(30):9020–9041.

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