The structural properties of magainin in water, TFE/water, and aqueous urea solutions: molecular dynamics simulations

Here, the MD simulations and comparative structural analysis of Magainin in water, TFE/water, and 2M, 4M, and BM urea solutions is reported. For MAG-TFE/water and MAG-2M urea the largely alpha helical conformation of the peptide is maintained throughout the 9-ns simulation. While in water, 4M urea, and 8M urea, the helix length decreases and at the same time helix radius increases. This suggests a more destabilized magainin secondary structure. Our simulation data reveals that the stabilizing effect of TFE is induced by preferential accumulation of TFE molecules around the alpha helical peptide. These results indicate that an aqueous urea solution solvates the surface of polypeptide chain more favorably than pure water. Urea molecules interact more favorably with nonpolar groups of the peptide in comparison with water, and the presence of urea improves the interactions of water molecules with the hydrophilic groups of the peptide. At 8M urea, there are more direct interactions between the urea and solute, and the helix is destabilized. At 2M urea, the interaction of urea molecules and nonpolar residues are weak, therefore, the presence of urea molecules decreases the interactions of water molecules with hydrophilic groups. Urea could not deteriorate the peptide secondary structure with time from an initial helix structure.     

Polyproline II helix is the preferred conformation for unfolded polyalanine in water

Does aqueous solvent discriminate among peptide conformers? To address this question, we computed the solvation free energy of a blocked, 12‐residue polyalanyl‐peptide in explicit water and analyzed its solvent structure. The peptide was modeled in each of 4 conformers: α‐helix, antiparallel β‐strand, parallel β‐strand, and polyproline II helix (P_II). Monte Carlo simulations in the canonical ensemble were performed at 300 K using the CHARMM 22 forcefield with TIP3P water. The simulations indicate that the solvation free energy of P_II is favored over that of other conformers for reasons that defy conventional explanation. Specifically, in these 4 conformers, an almost perfect correlation is found between a residue's solvent‐accessible surface area and the volume of its first solvent shell, but neither quantity is correlated with the observed differences in solvation free energy. Instead, solvation free energy tracks with the interaction energy between the peptide and its first‐shell water. An additional, previously unrecognized contribution involves the conformation‐dependent perturbation of first‐shell solvent organization. Unlike P_II, β‐strands induce formation of entropically disfavored peptide:water bridges that order vicinal water in a manner reminiscent of the hydrophobic effect. The use of explicit water allows us to capture and characterize these dynamic water bridges that form and dissolve during our simulations. Proteins 2004. © 2004 Wiley‐Liss, Inc.     

Conformational preferences of a short Aib/Ala‐based water‐soluble peptide as a function of temperature

The amino acid Aib predisposes a peptide to be helical with context‐dependent preference for either 3₁₀‐ or α‐ or a mixed helical conformation. Short peptides also show an inherent tendency to be unfolded. To characterize helical and unfolded states adopted by water‐soluble Aib‐containing peptides, the conformational preference of Ac‐Ala‐Aib‐Ala‐Lys‐Ala‐Aib‐Lys‐Ala‐Lys‐Ala‐Aib‐Tyr‐NH₂ was determined by CD, NMR and MD simulations as a function of temperature. Temperature‐dependent CD data indicated the contribution of two major components, each an admixture of helical and extended/polyproline II structures. Both right‐ and left‐handed helical conformations were detected from deconvolution of CD data and ¹³C NMR experiments. The presence of a helical backbone, more pronounced at the N‐terminal, and a temperature‐induced shift in α‐helix/3₁₀‐helix equilibrium, more pronounced at the C‐terminal, emerged from NMR data. Starting from polyproline II, the N‐terminal of the peptide folded into a helical backbone in MD simulations within 5 ns at 60°C. Longer simulations showed a mixed‐helical backbone to be stable over the entire peptide at 5°C while at 60°C the mixed‐helix was either stable at the N‐terminus or occurred in short stretches through out the peptide, along with a significant population of polyproline II. Our results point towards conformational heterogeneity of water‐soluble Aib‐based peptide helices and the associated subtleties. The problem of analyzing CD and NMR data of both left‐ and right‐handed helices are discussed, especially the validity of the ellipticity ratio [θ]₂₂₂/[θ]₂₀₇, as a reporter of α‐/3₁₀‐ population ratio, in right‐ and left‐handed helical mixtures. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Lipid-destabilising properties of a peptide with structural plasticity

The Chameleon peptide (Cham) is a peptide designed from two regions of the GB1 protein, one folded as an alpha-helix and the other as a beta structure. Depending on the environment, the Cham peptide adopts an alpha or a beta conformation when inserted in different locations of GB1. This environment dependence is also observed for tilted peptides. These short protein fragments, able to destabilise organised system, are mainly folded in beta structure in water and in alpha helix in a hydrophobic environment, like the lipid bilayer. In this paper, we tested whether the Cham peptide can be qualified as a tilted peptide. For this, we have compared the properties of Cham peptide (hydrophobicity, destabilising properties, conformation) to those of tilted peptides. The results suggest that Cham is a tilted peptide. Our study, together the presence of tilted fragments in transconformational proteins, suggests a relationship between tilted peptides and structural lability.     

A peptide study of the relationship between the collagen triple-helix and amyloid

Type XXV collagen, or collagen‐like amyloidogenic component, is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer's disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro‐Hyp‐Gly)₁₀, an amyloidogenic peptide GNNQQNY, and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)_n domains. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy showed the GNNQQNY peptide formed a random coil structure, whereas the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple‐helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, whereas amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple‐helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple‐helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly‐Xaa‐Yaa sequence and required the triple‐helix conformation. The inhibitory effect of the collagen triple‐helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests Type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 795–806, 2012.     

The part of a long beta hairpin from the scrapie form of the human prion protein is reconstructed in the synthetic CC36 protein

Mechanisms of beta sheet formation by the human prion protein are not clear yet. In this work, we clarified the role of the region containing C‐half of the second helix and N‐half of the third helix of that protein in the process of alpha helix to beta sheet transition. Solid phase automatic synthesis of the original peptide (CC36: Cys179–Cys214) failed because of the beta hairpin formation in the region 206‐MERVVEQMC‐214 with a high beta strand potential. Using Met206Arg and Val210Arg substitutions, we increased the probability of alpha helix formation by that sequence. After that modification, the complete CC36 peptide with disulfide bond has been synthesized. Modified peptide has been studied by circular dichroism (CD) and fluorescence spectrography. According to the CD spectra analysis, the CC36 peptide contains 37% of residues in beta sheet and just 15% in helix. Thermal analysis under the control of CD shows that the secondary structure content of the peptide is stable from 5°C to 80°C. Dissociation of oligomers of the CC36 peptide finishes at 37°C according to the fluorescence analysis. The CC36 peptide is able to bind Mn²⁺ cations, which causes small temperature‐associated structural shifts at concentrations of 2 – 10·10^?6 M. Predicted beta hairpin of the CC36 peptide (two beta strands are: 184‐IKQHTVT‐190 and 197‐TETDVKM‐205) should be the part of a longer beta hairpin from the scrapie form of the prion protein (PrPSc). Analogs of the CC36 peptide may be considered as antigens for the future development of a vaccine against PrPSc. Proteins 2016; 84:1462–1479. © 2016 Wiley Periodicals, Inc.     

A strained DNA binding helix is conserved for site recognition,folding nucleation,and conformational modulation

Nucleic acid recognition is often mediated by α‐helices or disordered regions that fold into α‐helix on binding. A peptide bearing the DNA recognition helix of HPV16 E2 displays type II polyproline (PII) structure as judged by pH, temperature, and solvent effects on the CD spectra. NMR experiments indicate that the canonical α‐helix is stabilized at the N‐terminus, while the PII forms at the C‐terminus half of the peptide. Re‐examination of the dihedral angles of the DNA binding helix in the crystal structure and analysis of the NMR chemical shift indexes confirm that the N‐terminus half is a canonical α‐helix, while the C‐terminal half adopts a 3₁₀ helix structure. These regions precisely match two locally driven folding nucleii, which partake in the native hydrophobic core and modulate a conformational switch in the DNA binding helix. The peptide shows only weak and unspecific residual DNA binding, 10⁴‐fold lower affinity, and 500‐fold lower discrimination capacity compared with the domain. Thus, the precise side chain conformation required for modulated and tight physiological binding by HPV E2 is largely determined by the noncanonical strained α‐helix conformation, “presented” by this unique architecture. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 432–443, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structural and dynamical studies of Humanin in water and TFE/water mixture: a molecular dynamics simulation

The structural and dynamical properties of Humanin, a small peptide with neuroprotective activity against the insults of the Alzheimer's disease-related genes and the neurotoxic amyloid peptide, are studied in two different environments by molecular dynamics simulation. In this study, we have performed comparative molecular dynamics simulations in the absence and in the presence of TFE. The resulting trajectories were analyzed in terms of structural and dynamical properties of peptide and compared to the available NMR data. In water humanin is observed to partly unfold. The peptide is readily stabilized in an ordered helical conformation in the TFE/water mixture. Our simulations show that the peptide is flexible with definite turn point in its structure in water environment. It is free to interact with receptors that mediate its action in polar environment. Humanin may also find an alpha helix structure necessary for passage through biomembranes and/or specific interactions.     

Helical formation of a 13-residue C-peptide analogue of ribonuclease A in sodium dodecyl sulfate solution

The conformation of a 13-residue C-peptide analogue of ribonuclease A——in surfactant solutions was studied by CD. The CD spectrum of the peptide in excess NaDodSO₄ solution was typical for a helical conformation; the spectrum appeared to be virtually independent of pH (2.5–6) and temperature (3–25°C). Analysis of the CD data indicated a helicity of about 65–70% with no α-sheet and β-turn; this corresponded to 8 or 9 residues in the helical form or slightly more than two turns of α-helix. This compares with an average of about one turn of α-helix for the C-peptide analogue in water at pH 4.7 and 7°C. The conformation of the peptide in cationic surfactant, dodecyl ammonium chloride, and nonionic surfactant, dodecyl heptaoxyethylene ether, solution resembled that in water. We concluded that the C-peptide analogue can develop a maximum helicity close to the corresponding segment in ribonuclease A in hydrophobic environment provided by the clustering of NaDodSO₄ molecules to the cationic side groups of the peptide, except that the end effects may destabilize two or three residues each at both ends of the helix. Thus, in the interior of a protein molecule this hydrophobic effect may overshadow the charged-group effect than can be explained by the helix dipole model for the helical segments on the exterior of the protein molecule.     

Two peptide fragments G55-I72 and K97-A109 from staphylococcal nuclease exhibit different behaviors in conformational preferences for helix formation

Two synthetic peptides, SNasealpha1 and SNasealpha2, corresponding to residues G55-I72 and K97-A109, respectively, of staphylococcal nuclease (SNase), are adopted for detecting the role of helix alpha1 (E57-A69) and helix alpha2 (M98-Q106) in the initiation of folding of SNase. The helix-forming tendencies of the two SNase peptide fragments are investigated using circular dichroism (CD) and two-dimensional (2D) nuclear magnetic resonance (NMR) methods in water and 40% trifluoroethanol (TFE) solutions. The coil-helix conformational transitions of the two peptides in the TFE-H2O mixture are different from each other. SNasealpha1 adopts a low population of localized helical conformation in water, and shows a gradual transition to helical conformation with increasing concentrations of TFE. SNasealpha2 is essentially unstructured in water, but undergoes a cooperative transition to a predominantly helical conformation at high TFE concentrations. Using the NMR data obtained in the presence of 40% TFE, an ensemble of alpha-helical structures has been calculated for both peptides in the absence of tertiary interactions. Analysis of all the experimental data available indicates that formation of ordered alpha-helical structures in the segments E57-A69 and M98-Q106 of SNase may require nonlocal interactions through transient contact with hydrophobic residues in other parts of the protein to stabilize the helical conformations in the folding. The folding of helix alpha1 is supposed to be effective in initiating protein folding. The formation of helix alpha2 depends strongly on the hydrophobic environment created in the protein folding, and is more important in the stabilization of the tertiary conformation of SNase.     

Alpha helical crossovers favor right‐handed supersecondary structures by kinetic trapping: The phone cord effect in protein folding

The remarkable predominance of right‐handedness in beta‐alpha‐beta helical crossovers has been previously explained in terms of thermodynamic stability and kinetic accessibility, but a different kinetic trapping mechanism may also play a role. If the beta‐sheet contacts are made before the crossover helix is fully formed, and if the backbone angles of the folding helix follows the energetic pathway of least resistance, then the helix would impart a torque on the ends of the two strands. Such a torque would tear apart a left‐handed conformation but hold together a right‐handed one. Right‐handed helical crossovers predominate even in all‐alpha proteins, where previous explanations based on the preferred twist of the beta sheet do not apply. Using simple molecular simulations, we can reproduce the right‐handed preference in beta‐alpha‐beta units, without imposing specific beta‐strand geometry. The new kinetic trapping mechanism is dubbed the “phone cord effect” because it is reminiscent of the way a helical phone cord forms superhelices to relieve torsional stress. Kinetic trapping explains the presence of a right‐handed superhelical preference in alpha helical crossovers and provides a possible folding mechanism for knotted proteins.     

High‐resolution structures of collagen‐like peptides [(Pro‐Pro‐Gly)4‐Xaa‐Yaa‐Gly‐(Pro‐Pro‐Gly)4]: Implications for triple‐helix hydration and Hyp(X) puckering

Structures of (Pro‐Pro‐Gly)₄‐Xaa‐Yaa‐Gly‐(Pro‐Pro‐Gly)₄ (ppg9‐XYG) where (Xaa, Yaa) = (Pro, Hyp), (Hyp, Pro) or (Hyp, Hyp) were analyzed at high resolution using synchrotron radiation. Molecular and crystal structures of these peptides are very similar to those of the (Pro‐Pro‐Gly)₉ peptide. The results obtained in this study, together with those obtained from related compounds, indicated the puckering propensity of the Hyp in the X position: (1) Hyp(X) residues involved in the Hyp(X):Pro(Y) stacking pairs prefer the down‐puckering conformation, as in ppg9‐OPG, and ppg9‐OOG; (2) Hyp(X) residues involved in the Hyp(X):Hyp(Y) stacking pairs prefer the up‐puckering conformation if there is no specific reason to adopt the down‐puckering conformation. Water molecules in these peptide crystals are classified into two groups, the 1st and 2nd hydration waters. Water molecules in the 1st hydration group have direct hydrogen bonds with peptide oxygen atoms, whereas those in the 2nd hydration group do not. Compared with globular proteins, the number of water molecules in the 2nd hydration shell of the ppg9‐XYG peptides is very large, likely due to the unique rod‐like molecular structure of collagen model peptides. In the collagen helix, the amino acid residues in the X and Y positions must protrude outside of the triple helix, which forces even the hydrophobic side chains, such as Pro, to be exposed to the surrounding water molecules. Therefore, most of the waters in the 2nd hydration shell are covering hydrophobic Pro side chains by forming clathrate structures. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 361–372, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Inter-chain Proline: Proline Contacts Contribute to the Stability of the Triple Helical Conformation

Abstract

The triple helical conformation observed in the collagen group of proteins is related to the presence of large numbers of imino residues and is derived from the stereochemical properties of these residues. The triple helix is stabilized by increasing numbers of these residues. Hydrogen bonds are usually considered to be a major factor in the formation and stability of protein conformation, however, imino residues are not hydrogen bond donors. We have evaluated the role of these residues in stabilizing the triple helix by re-examining two X-ray based structures of the triple helical polypeptide (Pro-Pro- Gly)₁₀ using molecular mechanics calculations. The two minimized structures are comparable in energy and have helical parameters close to the starting values for each starting structure. Our studies suggest that clusters of close van der Waals contacts between proline residues in adjacent chains contribute significantly to the stability of the triple helix. Preliminary NMR studies support this concept. We propose that non-bonded interactions between proline residues may be a significant stabilizing force in the triple helix generated by (Pro-Pro-Gly)₁₀.     

Specific interactions of sticholysin I with model membranes: An NMR study

Sticholysin I (StnI) is an actinoporin produced by the sea anemone Stichodactyla helianthus that binds biological and model membranes forming oligomeric pores. Both a surface cluster of aromatic rings and the N‐terminal region are involved in pore formation. To characterize the membrane binding by StnI, we have studied by ¹H‐NMR the environment of these regions in water and in the presence of membrane‐mimicking micelles. Unlike other peptides from homologous actinoporins, the synthetic peptide corresponding to residues 1–30 tends to form helix in water and is more helical in either trifluoroethanol or dodecylphosphocholine (DPC) micelles. In these environments, it forms a helix‐turn‐helix motif with the last α‐helical segment matching the native helix‐α₁ (residues 14–24) present in the complete protein. The first helix (residues 4–9) is less populated and is not present in the water‐soluble protein structure. The characterization of wild‐type StnI structure in micelles shows that the helix‐α₁ is maintained in its native structure and that this micellar environment does not provoke its detachment from the protein core. Finally, the study of the aromatic resonances has shown that the motional flexibility of specific rings is perturbed in the presence of micelles. On these bases, the implication of the aromatic rings of Trp‐111, Tyr‐112, Trp‐115, Tyr‐132, Tyr‐136, and Tyr‐137, in the interaction between StnI and the micelle is discussed. Based on all the findings, a revised model for StnI interaction with membranes is proposed, which accounts for differences in its behavior as compared with other highly homologous sticholysins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Design and synthesis of sequence-specific DNA-binding peptides

Design, synthesis and DNA binding activities of two peptides containing 32 and 102 residues are reported. A nonlinear 102-residue peptide contains four modified alpha helix-turn-alpha helix motifs of 434 cro protein. These four units are linked covalently to a carboxyterminal crosslinker containing four arms each ending with an aliphatic amino group. From CD studies we have found that in aqueous buffer in the presence of 20% trifluoroethanol the peptide residues assume alpha-helical, beta-sheet and random-coiled conformations with the alpha-helical content of about 16% at room temperature. Upon complex formation between peptide and DNA, a change in the peptide conformation takes place which is consistent with an alpha - beta transition in the DNA binding alpha helix-turn-alpha helix units of the peptide. Similar conformation changes are observed upon complex formation with the synthetic operator of a linear peptide containing residues 7-37 of 434 cro repressor. Evidently, in the complex, residues present in helices alpha 2 and alpha 3 of the two helix motif form a beta-hairpin which is inserted in the minor DNA groove. The last inference is supported by our observations that the two peptides can displace the minor groove-binding antibiotic distamycin A from poly(dA).poly(dT) and synthetic operator DNA. As revealed from DNase digestion studies, the nonlinear peptide binds more strongly to a pseudooperator Op1, located in the cro gene, than to the operator OR3. A difference in the specificity shown by the non-linear peptide and wild-type cro could be attributed to a flexibility of the linker chains between the DNA-binding domains in the peptide molecule as well as to a replacement of Thr-Ala in the peptide alpha 2-helices. Removal of two residues from the N-terminus of helix alpha 2 in each of the four DNA-binding domains of the peptide leads to a loss of binding specificity.     

Correlation between computed conformational properties of cytochrome c peptides and their antigenicity in a T-lymphocyte proliferation assay

By means of conformational energy calculations, we previously showed that the antigenic strength of a series of oligopeptides (derived from the carboxyl terminal sequence of cytochrome c) in a T-lymphocyte proliferation assay depends on their ability to adopt the α-helix conformation. Using experimentally determined statistical weights (within the framework of the Zimm–Bragg theory for the helix–coil transition), here we present a simple free energy analysis of the ability of these peptides to adopt the α-helix conformation in water. The experimental statistical weights have been modified to include the effect of long-range charge–dipole interactions on helix stability. We find that there is a close correlation between the tendency of a peptide to adopt the α-helix conformation and its ability to stimulate antigen-primed T cells. The shortest peptide with a tendency to adopt the α-helix conformation is also the shortest one that exhibits antigenic activity. The rapid and simple method presented here can thus be used to predict relative antigenicities for different peptides derived from cytochrome c.     

Single-residue substitution in homopolypeptides: Perturbative helix–coil theory at a single site

Based on Lifson–Roig's helix–coil transition theory, substitution of a single heteroresidue into a homopolymer host is studied. This study models recent experiments that substitute a single amino acid into a small peptide in water [A. Chakrabartty, J. A. Schellman, and R. L. Baldwin (1991), Nature, Vol. 351, pp. 586–688]. Our formalism, which is based on a perturbation method, differs from the existing theory for sequenced polymers and is naturally analogous, hence likely to be useful, to substitution experiments in the laboratory. It is shown that the intrinsic helix propensity w is directly proportional to the equilibrium constant for the helix–coil equilibrium of a single residue in a host peptide. This intuitive new result will simplify experimental data interpretations for measurements of the helical conformation on the single amino acid level. It is also shown that substitution affects the total helicity of the host peptide according to two considerations: the helicity of the replaced residue prior to the substitution, and the sensitivity of the site, a measure of neighboring interactions. The relationship between substitution stability and thermal stability is explored. © 1993 John Wiley & Sons, Inc.     

Conserved water molecules in MHC class-I molecules and their putative structural and functional roles

A set of conserved water positions making direct contacts with the alpha1 and alpha2 domains of the MHC class-I protein was identified by a cluster analysis in 12 high-resolution crystal structures of proteins from different allele types and different species, comprising human, mouse and rat. The analysis revealed a total of 63 clusters, corresponding to water molecules, whose positions are conserved in half or more of the analyzed structures. Analysis of these clusters shows that the most conserved water positions-those appearing in the largest fraction of the structures-were also the most accurately defined, as measured by their normalized crystallographic B-factor. Not too surprisingly, these positions displayed better overlap and formed more H-bonds with the protein. In a second part of this work, a detailed analysis is presented of three of the most conserved water positions and their putative structural and functional roles are discussed. The most highly conserved of the three appears to play an important role in stabilizing the conformation of a twisted beta-turn between residues 118 and 122 (numbering of HLA-B3501, PDB code 1A1N). An equivalent water molecule was found to be associated with a similar beta-turn in 43 unrelated structures surveyed in the PDB, leading to the suggestion that this water molecule plays an important structural role in this type of turn. The second water molecule makes hydrogen bonds with residues lining pocket B in the peptide-binding groove and is suggested to play a role in modulating peptide recognition. The third highly conserved water molecule is located at the first kink of the alpha2 helix, possibly playing a role in determining the position of the N-terminal segment of that helix, which also carries side chains in contact with the bound peptide. This information on conserved water positions in MHC class-I molecules should be helpful in modeling interactions with bound peptide antigens and in designing new peptides with tailor-made affinities.     

Dissection of the de novo designed peptide alpha t alpha: stability and properties of the intact molecule and its constituent helices

Alpha t alpha is a de novo designed 38-residue peptide [Fezoui et al. (1995) Protein Sci. 4, 286-295] that adopts a helical hairpin conformation in solution [Fezoui et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3675-3679; Fezoui et al. (1997) Protein Sci. 6, 1869-1877]. Since alpha t alpha was developed as a model system for protein folding at the stage where secondary structures interact and become mutually stabilizing, it is of interest to investigate the increase in stability that occurs with helix association. alpha t alpha was dissected into its component helices and the relative stabilities of the individual helices and the parent molecule were assessed. The Delta G0 of unfolding of alpha t alpha measured by guanidine hydrochloride denaturation was determined to be 3.4 kcal/mol. The equilibrium constant for folding of alpha t alpha was estimated from the Delta G0 as 338 and from hydrogen exchange measurements as 259. The stability of the helices in intact alpha t alpha over the individual helices increased by a factor of at least 37 based on amide proton exchange measurements. Sedimentation equilibrium studies showed very little association of the peptides to form either homo- or heterodimers, suggesting that helix association is stabilized by the high effective concentration of the helices caused by the presence of the connecting turn. The effects of salt and pH on the helicity of the component peptides are largely reflected in the intact molecule, implying that short-range interactions still make important contributions to the conformation of the intact molecule even though significant stabilization is caused by helix association.