Water-Mediated Electronic Structure of Oligopeptides Probed by Their UV Circular Dichroism, Absorption Spectra, and Time-Dependent DFT Calculations.

We investigate the UV absorption spectra of a series of cationic GxG peptides (where x denotes a guest residue) in aqueous solution and find that only a subset of these spectra show a strong dependence with temperature. To explore whether or not this observation reflects conformational dependencies, we carry out time-dependent density functional calculations for the polyproline II (pPII) and ß-strand conformations in implicit and explicit water. We find that the calculated CD spectra for pPII can qualitatively account for the experimental spectra irrespective of the water model. The ß-strand UV-CD spectra, however, require the explicit consideration of water. Contrary to conventional wisdom, we find that both the NV1 and NV2 band are the envelopes of contributions from multiple transitions that involve more than just the HOMOs and LUMOs of the peptide groups. A natural transition orbital analysis reveals that some of the transitions have a charge-transfer character. The overall manifold of transitions depends on the peptide's backbone conformation, peptide hydration, and side chain of the guest residue. Our results reveal that peptide groups, side chains, and hydration shells must be considered as an entity for a physically valid characterization of UV absorbance and circular dichroism.

Anticooperative Nearest-Neighbor Interactions between Residues in Unfolded Peptides and Proteins.

Growing evidence suggests that the conformational distributions of amino acid residues in unfolded peptides and proteins depend on the nature of the nearest neighbors. To explore whether the underlying interactions would lead to a breakdown of the isolated pair hypothesis of the classical random coil model, we further analyzed the conformational propensities that were recently obtained for the two guest residues (x,y) of GxyG tetrapeptides. We constructed a statistical thermodynamics model that allows for cooperative as well as for anticooperative interactions between adjacent residues adopting either a polyproline II or a ß-strand conformation. Our analysis reveals that the nearest-neighbor interactions between most of the central residues in the investigated GxyG peptides are anticooperative. Interaction Gibbs energies are rather large at high temperatures (350 K), at which point many proteins undergo thermal unfolding. At room temperature, these interaction energies are less pronounced. We used the obtained interaction parameter in a Zimm-Bragg/Ising-type approach to calculate the temperature dependence of the ultraviolet circular dichroism (CD) of the MAX3 peptide, which is predominantly built by KV repeats. The agreement between simulation and experimental data was found to be satisfactory. Finally, we analyzed the temperature dependence of the CD and 3J(HNHα) parameters of the amyloid ß1-9 fragment. The results of this analysis and a more qualitative consideration of the temperature dependence of denatured proteins probed by CD spectroscopy further corroborate the dominance of anticooperative nearest-neighbor interactions. Generally, our results show that unfolded peptides-and most likely also proteins-exhibit some similarity with antiferromagnetic systems.

Investigating the Formation of a Repulsive Hydrogel of a Cationic 16mer Peptide at Low Ionic Strength in Water by Vibrational Spectroscopy and Rheology.

The cationic peptide (AAKA)4 (AK16) exhibits a high propensity for aggregation into ß-sheet-like structures in spite of the high positive charge of its protonated lysine side chains. Upon incubation into an aqueous solution, the peptide maintains a metastable ß-sheet-like structure with fibrillar content, the apparent stability of which increases with peptide concentration. In the presence of a sufficiently high concentration of anions, the peptide spontaneously forms a hydrogel at millimolar concentrations. Interestingly, we find that even in the absence of gel-supporting anions, the peptide is capable of forming a hydrogel in the centimolar range. Rheological data reveal that the gel is a stable elastic solid. These data show that the peptide can overcome the repulsive interactions between the positively charged ammonium groups of the lysine residues. The addition of 1 M NaCl just accelerates this process. Atomic force microscopy images of the peptide gel reveal fibrils with thicknesses between 4 and 8 nm, which suggests that they contain multiple layers of sheets. We propose that long tapes of ß-sheet are arranged in fibrils via stacking of alternating interfaces induced by hydrophobic interactions between alanine side chains and by the formation of a hydrogen bonded water network between hydrophilic sides of AK16 ß-sheets, which leads to the observed immobilization of the solvent in the formed hydrogel. Water immobilization is proposed as the likely cause for a significant increase in the amide I' oscillator strength of the formed ß-sheet structures.

Construction and comparison of the statistical coil states of unfolded and intrinsically disordered proteins from nearest-neighbor corrected conformational propensities of short peptides.

Assessing the influence of nearest neighbors on the conformational ensemble of amino acid residues in unfolded and intrinsically disordered proteins and peptides is pivotal for a thorough understanding of the statistical coil state of unfolded proteins as well as of the energetics of the folding process. Research aimed at exploring nearest neighbor interactions has mostly focused on the analysis of restricted coil libraries that reflect conformational distributions in loops connecting more regular secondary structure segments. Recently, however, Toal et al. reported an experimentally based structural analysis of selected xy-pairs in GxyG tetrapeptides, which revealed quantitative information about conformational changes induced by nearest-neighbor interactions (Eur. J. Chem., 2015, 21, 5173-5192). Here, we perform analyses of Ramachandran plots of xy-pairs in GxyG and in coil libraries (Ting et al., PLOS CompBiol, 2010, 6, e1000763) using Hellinger distances as a quantitative measure of dissimilarities between Ramachandran distributions. Our analysis reveals that nearest-neighbor effects inferred from the above coil library are much less pronounced than corresponding structural changes observed for GxyG peptides. To determine whether nearest-neighbor induced conformational changes observed for GxyG can be utilized for the analysis of unfolded proteins, we analyzed sets of 3J(HHHα) coupling constants of three different unfolded proteins, namely the 130-residue fragment of the Staphylococcus aureus fibronectin-binding protein (FnBPc), denatured hen lysozyme, and the htau40 protein. For the first two proteins we found statistically meaningful correlations between predicted nearest-neighbor induced changes of 3J(HHHα) and experimentally observed deviations from corresponding coupling constants of GxG peptides in water, which we used as reference system with minimal nearest-neighbor interactions. This observation is in line with the NMR based understanding of these proteins being predominantly statistical coils. For htau40, however, which is known to exhibit residual structure and large deviations form statistical coil expectations, these correlations are weak or absent. Our results thus underscore the importance of nearest-neighbor interactions for a complete physical description of an ideal statistical coil state of a protein.

Water-Centered Interpretation of Intrinsic pPII Propensities of Amino Acid Residues: In Vitro-Driven Molecular Dynamics Study.

Amino acid residues of unfolded peptides in water sample only a few basins in the Ramachandran plot, including prominent polyproline II-like (pPII) conformations. Dynamics of guest residues, X, in GXG peptides in water were recently reported to be dominated by pPII and ß-strand-like (ß) conformations, resulting in an enthalpy-entropy compensation at ∼300 K. Using molecular dynamics (MD) in explicit solvent, we here examine pPII and ß conformational ensembles of 15 guest residues in GXG peptides, quantify local orientation of water around their side chains through novel water orientation plots, and study their hydration and hydrogen bonding properties. We show that pPII and ß ensembles are characterized by distinct water orientations: pPII ensembles are associated with an increased population of water oriented in parallel to the side chain surface whereas ß ensembles exhibit more heterogeneous water orientations. The backbone hydration is significantly higher in pPII than in ß ensembles. Importantly, pPII to ß hydration differences and the solvent accessible surface area of Cß hydrogens both correlate with experimental pPII propensities. We propose that pPII conformations are stabilized by a local, hydrogen-bonded clathrate-like water structure and that residue-specific intrinsic pPII propensities reflect distinct abilities of side chains to template this water structure.

Demixing of water and ethanol causes conformational redistribution and gelation of the cationic GAG tripeptide.

The cationic tripeptide GAG undergoes three conformational changes in binary mixtures of water and ethanol. At 17 mol% of ethanol conformational sampling is shifted from pPII towards ß-strands. A more pronounced shift in the same direction occurs at 40 mol%. At ca. 55 mol% of ethanol and above a peptide concentration of ca. 0.2 M the ternary peptide-water-ethanol mixture forms a hydrogel which is comprised of unusually large crystalline like non-ß sheet fibrils forming a sample spanning matrix.

Assessing backbone solvation effects in the conformational propensities of amino acid residues in unfolded peptides.

Conformational ensembles of individual amino acid residues within model GxG peptides (x representing different amino acid residues) are dominated by a mixture of polyproline II (pPII) and ß-strand like conformations. We recently discovered rather substantial differences between the enthalpic and entropic contributions to this equilibrium for different amino acid residues. Isoleucine and valine exceed all other amino acid residues in terms of their rather large enthalpic stabilization and entropic destabilization of polyproline II. In order to shed light on these underlying physical mechanisms, we performed high-level DFT calculations to explore the energetics of four representative GxG peptides where x = alanine (A), leucine (L), valine (V), and isoleucine (I) in explicit water (10 H2O molecules with a polarizable continuum water model) and in vacuo. We found that the large energetic contributions to the stabilization of pPII result, to a major extent, from peptide-water, water-water interactions, and changes of the solvent self-energy. Differences between the peptide-solvent interaction energies of hydration in pPII and ß-strand peptides are particularly important for the pPII ⇌ ß equilibria of the more aliphatic peptides GIG and GLG. Furthermore, we performed a vibrational analysis of the four peptides in both conformations and discovered a rather substantial mixing between water motions and peptide vibrations below 700 cm(-1). We found that the respective vibrational entropies are substantially different for the considered conformations, and their contributions to the Gibbs/Helmholtz energy stabilize ß-strand conformations. Taken together, our results underscore the notion of the solvent being the predominant determinant of peptide (and protein) conformations in the unfolded state.

Randomizing the unfolded state of peptides (and proteins) by nearest neighbor interactions between unlike residues.

To explore the influence of nearest neighbors on conformational biases in unfolded peptides, we combined vibrational and 2D NMR spectroscopy to obtain the conformational distributions of selected "GxyG" host-guest peptides in aqueous solution: GDyG, GSyG, GxLG, GxVG, where x/y=A, K, L, V. Large changes of conformational propensities were observed due to nearest-neighbor interactions, at variance with the isolated pair hypothesis. We found that protonated aspartic acid and serine lose their above-the-average preference for turn-like structures in favor of polyproline II (pPII) populations in the presence of neighbors with bulky side chains. Such residues also decrease the above-the-average pPII preference of alanine. These observations suggest that the underlying mechanism involves a disruption of the hydration shell. Thermodynamic analysis of (3) J(H(N) ,H(α) ) (T) data for each x,y residue reveals that modest changes in the conformational ensemble masks larger changes of enthalpy and entropy governing the pPIIâ†”ß equilibrium indicating a significant residue dependent temperature dependence of the peptides' conformational ensembles. These results suggest that nearest-neighbor interactions between unlike residues act as conformational randomizers close to the enthalpy-entropy compensation temperature, eliminating intrinsic biases in favor of largely balanced pPII/ß dominated ensembles at physiological temperatures.

Entropy reduction in unfolded peptides (and proteins) due to conformational preferences of amino acid residues.

As established by several groups over the last 20 years, amino acid residues in unfolded peptides and proteins do not exhibit the unspecific random distribution as assumed by the classical random coil model. Individual amino acid residues in small peptides were found to exhibit different conformational preferences. Here, we utilize recently obtained conformational distributions of guest amino acid residues in GxG peptides to estimate their conformational entropy, which we find to be significantly lower than the entropy of an assumed random coil like distribution. Only at high temperature do backbone entropies approach random coil like values. We utilized the obtained backbone entropies of the investigated amino acid residues to estimate the loss of conformational entropy caused by a coil → helix transition and identified two subsets of amino acid residues for which the thus calculated entropy losses correlate well with the respective Gibbs energy of helix formation obtained for alanine based host-guest systems. Calculated and experimentally derived entropic losses were found to be in good agreement. For most of the amino acid residues investigated entropic losses derived from our GxG distributions correlate very well with corresponding values recently obtained from MD simulations biased by conformational propensities derived from truncated coil libraries. Both, conformational entropy and the entropy of solvation exhibit a strong, residue specific temperature dependence, which can be expected to substantially affect the stability of unfolded states. Altogether, our results provide strong evidence for the notion that conformational preferences of amino acid residues matter with regard to the thermodynamics of peptide and protein folding.

Local order in the unfolded state: conformational biases and nearest neighbor interactions.

The discovery of Intrinsically Disordered Proteins, which contain significant levels of disorder yet perform complex biologically functions, as well as unwanted aggregation, has motivated numerous experimental and theoretical studies aimed at describing residue-level conformational ensembles. Multiple lines of evidence gathered over the last 15 years strongly suggest that amino acids residues display unique and restricted conformational preferences in the unfolded state of peptides and proteins, contrary to one of the basic assumptions of the canonical random coil model. To fully understand residue level order/disorder, however, one has to gain a quantitative, experimentally based picture of conformational distributions and to determine the physical basis underlying residue-level conformational biases. Here, we review the experimental, computational and bioinformatic evidence for conformational preferences of amino acid residues in (mostly short) peptides that can be utilized as suitable model systems for unfolded states of peptides and proteins. In this context particular attention is paid to the alleged high polyproline II preference of alanine. We discuss how these conformational propensities may be modulated by peptide solvent interactions and so called nearest-neighbor interactions. The relevance of conformational propensities for the protein folding problem and the understanding of IDPs is briefly discussed.

The TrpA protein of Trichodesmium erythraeum IMS101 is a non-fibril-forming collagen and a component of the outer sheath.

Collagen molecules are structural in nature and primarily found in eukaryotic, multicellular organisms. Recently, a collagen-like protein, TrpA, was identified and characterized in the marine cyanobacterium Trichodesmium erythraeum IMS 101, and it was shown to be involved in maintaining the structural integrity of the trichomes. The TrpA protein contains one glycine interruption in the otherwise perfectly uninterrupted collagenous domain. In this study, we used phylogenetic analysis to determine that the TrpA protein sequence is most closely associated with non-fibril-forming collagen proteins. Structural modelling and circular dichroism data suggest that the glycine insertion decreases the stability of TrpA compared to uninterrupted collagen sequences. Additionally, scanning electron microscopy revealed that TrpA is expressed entirely on the surface of the trichomes, with no specific pattern of localization. These data indicate that the TrpA protein is part of the outer sheath of this organism. As such, this protein may function to promote adhesion between individual T. erythraeum trichomes, and between this organism and heterotrophic bacteria found in the same environment.

Role of enthalpy-entropy compensation interactions in determining the conformational propensities of amino acid residues in unfolded peptides.

The driving forces governing the unique and restricted conformational preferences of amino acid residues in the unfolded state are still not well understood. In this study, we experimentally determine the individual thermodynamic components underlying intrinsic conformational propensities of these residues. Thermodynamic analysis of ultraviolet-circular dichroism (UV-CD) and (1)H NMR data for a series of glycine capped amino acid residues (i.e., G-x-G peptides) reveals the existence of a nearly exact enthalpy-entropy compensation for the polyproline II-ß strand equilibrium for all investigated residues. The respective ΔHß, ΔSß values exhibit a nearly perfect linear relationship with an apparent compensation temperature of 295 ± 2 K. Moreover, we identified iso-equilibrium points for two subsets of residues at 297 and 305 K. Thus, our data suggest that within this temperature regime, which is only slightly below physiological temperatures, the conformational ensembles of amino acid residues in the unfolded state differ solely with respect to their capability to adopt turn-like conformations. Such iso-equilibria are rarely observed, and their existence herein indicates a common physical origin behind conformational preferences, which we are able to assign to side-chain dependent backbone solvation. Conformational effects such as differences between the number of sterically allowed side chain rotamers can contribute to enthalpy and entropy but not to the Gibbs energy associated with conformational preferences. Interestingly, we found that alanine, aspartic acid, and threonine are the only residues which do not share these iso-equilbiria. The enthalpy-entropy compensation discovered as well as the iso-equilbrium and thermodynamics obtained for each amino acid residue provide a new and informative way of identifying the determinants of amino acid propensities in unfolded and disordered states.

pH-Independence of trialanine and the effects of termini blocking in short peptides: a combined vibrational, NMR, UVCD, and molecular dynamics study.

Several lines of evidence now well establish that unfolded peptides in general, and alanine in specific, have an intrinsic preference for the polyproline II (pPII) conformation. Investigation of local order in the unfolded state is, however, complicated by experimental limitations and the inherent dynamics of the system, which has in some cases yielded inconsistent results from different types of experiments. One method of studying these systems is the use of short model peptides, and specifically short alanine peptides, known for predominantly sampling pPII structure in aqueous solution. Recently, He et al. ( J. Am. Chem. Soc. 2012 , 134 , 1571 - 1576 ) proposed that unblocked tripeptides may not be suitable models for studying conformational propensities in unfolded peptides due to the presence of end effect, that is, electrostatic interactions between investigated amino acid residues and terminal charges. To determine whether changing the protonation states of the N- and C-termini influence the conformational manifold of the central amino acid residue in tripeptides, we have examined the pH-dependence of unblocked trialanine and the conformational preferences of alanine in the alanine dipeptide. To this end, we measured and globally analyzed amide I' band profiles and NMR J-coupling constants. We described conformational distributions as the superposition of two-dimensional Gaussian distributions assignable to specific subspaces of the Ramachandran plot. Results show that the conformational ensemble of trialanine as a whole, and the pPII content (χpPII = 0.84) in particular, remains practically unaffected by changing the protonation state. We found that compared to trialanine, the alanine dipeptide has slightly lower pPII content (χpPII = 0.74) and an ensemble more reminiscent of the unblocked Gly-Ala-Gly model peptide. In addition, a two-state thermodynamic analysis of the conformational sensitive Δε(T) and (3)J(H(N)H(α))(T) data obtained from electronic circular dichroism and H NMR spectra indicate that the free energy landscape of trialanine is similar in all protonation states. MD simulations for the investigated peptides corroborate this notion and show further that the hydration shell around unblocked trialanine is unaffected by the protonation/deprotonation of the C-terminal group. In contrast, the alanine dipeptide shows a reduced water density around the central residue as well as a less ordered hydration shell, which decreases the pPII propensity and reduces the lifetime of sampled conformations.

Disorder and order in unfolded and disordered peptides and proteins: a view derived from tripeptide conformational analysis. I. Tripeptides with long and predominantly hydrophobic side chains.

We performed a conformational analysis of the central residues of three tripeptides glycyl-L-isoleucyl-glycine (GIG), glycyl-L-tyrosyl-glycine (GYG) and glycyl-L-arginyl-glycine (GRG) in aqueous solution, based on a global analysis of amide I' band profiles and NMR J-coupling constants. The results are compared with recently reported distributions of GVG, GFG and GEG. For GIG and GYG, we found that even though the polyproline II (pPII) fraction is below 0.5, it is still the most populated conformation, whereas GVG and GFG show both a larger ß-strand fraction. For GRG, we observed a clear dominance of pPII over ß-strand, reminiscent of observations for GEG and GKG. This finding indicates that terminal charges on otherwise hydrophobic residue side chains stabilize pPII over ß-strand conformations. For all peptides investigated we found that a variety of compact and turn-like conformations constitute nearly 20 percent of their conformational distributions. Attempts to analyze our data with a simple two-state pPII-->/<--ß model therefore do not yield any satisfactory reproduction of experimental results. A comparison of the obtained GxG ensembles with conformational distributions of GxG segments in truncated coil libraries (helices and sheets omitted) revealed a much larger fraction of type II ß(i+2) and type III ß like conformations for the latter. Thus, a comparison of conformational distributions of unfolded peptide segments in solution and in coil libraries reveal interesting information on how the interplay between intrinsic propensities of amino acid residues and non-local interactions in polypeptide chains determine the conformations of loop segments in proteins.

Disorder and order in unfolded and disordered peptides and proteins: a view derived from tripeptide conformational analysis. II. Tripeptides with short side chains populating asx and ß-type like turn conformations.

In the preceding paper, we found that ensembles of tripeptides with long or bulky chains can include up to 20% of various turns. Here, we determine the structural and thermodynamic characteristics of GxG peptides with short polar and/or ionizable central residues (D, N, C), whose conformational distributions exhibit higher than average percentage (>20%) of turn conformations. To probe the side-chain conformations of these peptides, we determined the (3)J(H(α),H(ß)) coupling constants and derived the population of three rotamers with χ1 -angles of -60°, 180° and 60°, which were correlated with residue propensities by DFT-calculations. For protonated GDG, the rotamer distribution provides additional evidence for asx-turns. A comparison of vibrational spectra and NMR coupling constants of protonated GDG, ionized GDG, and the protonated aspartic acid dipeptide revealed that side chain protonation increases the pPII content at the expense of turn populations. The charged terminal groups, however, have negligible influence on the conformational properties of the central residue. Like protonated GDG, cationic GCG samples asx-turns to a significant extent. The temperature dependence of the UVCD spectra and (3)J(H(N)H(α)) constants suggest that the turn populations of GDG and GNG are practically temperature-independent, indicating enthalpic and entropic stabilization. The temperature-independent J-coupling and UVCD spectra of GNG require a three-state model. Our results indicate that short side chains with hydrogen bonding capability in GxG segments of proteins may serve as hinge regions for establishing compact structures of unfolded proteins and peptides.

Structural analysis of unfolded peptides by Raman spectroscopy.

Raman spectroscopy has positioned itself as an invaluable tool in the study of complex biological systems, consistently being used to obtain information illustrating a vast array of fundamental properties. Of primary interest, with respect to the focus of this chapter, are conformational changes of peptide backbones. For short peptides to larger biological systems this understanding can be extended to local hydrogen bonding interactions and the probing of other structural or organizational properties. With regard to unfolded peptides Raman spectroscopy can be used as a technique complementary to infrared (IR) and vibrational circular dichroism (VCD) spectroscopy. This chapter describes how high quality polarized Raman spectra of peptide can be recorded with a Raman microspectrometer and how the structure sensitive amide I band profiles of isotropic and anisotropic Raman scattering can be analyzed in conjunction with the respective IR and VCD profiles to obtain conformational distributions of short unfolded peptides.

Ionized trilysine: a model system for understanding the nonrandom structure of poly-L-lysine and lysine-containing motifs in proteins.

It is now well-established that different amino acid residues can exhibit different conformational distributions in the unfolded state of peptides and proteins. These conformational propensities can be modulated by nearest neighbors. In the current study, we combined vibrational and NMR spectroscopy to determine the conformational distributions of the central and C-terminal residues in trilysine peptides in aqueous solution. The study was motivated by earlier observations suggesting that interactions between ionized nearest neighbor residues can substantially change conformational propensities. We found that the central lysine residue predominantly adopts conformations that are located at the upper border of the upper left quadrant of the Ramachandran plot and the left border of the polyproline II region. We term this type of conformation deformed polyproline II (pPII(d)). The structures of less populated subensembles of trilysine resemble are comparable with structures at the i + 1 position of type I and type II ß-turns. For the C-terminal residue, however, we obtained a mixture of polyproline II, ß-strand, and right-handed helical conformations, which is typical for lysine residues in alanine- and glycine-based peptides. Our data thus indicate that the terminal lysines modify and restrict the conformational distribution of the central lysine residue. DFT calculations for ionized trilysine and lysyllysyllysylglycine in vacuo indicate that the pPII(d) is stabilized by a rather strong hydrogen bond between the NH3(+) group of the central lysine and the carbonyl group of the C-terminal peptide. This intramolecular hydrogen bonding induces optical activity in the C-terminal CO stretching vibration, which leads to an unusual and relatively intense positive Cotton band. Additionally, we analyzed the amide I' band profile of ionized triornithine in water. Ornithine is structurally similar to lysine in that its side chain is terminated with an amino group; however, the side chain of ornithine is shorter than lysine's side chain by one methylene group. We found that the conformational distribution of the central ornithine in this peptide must be very similar to that of the central lysine residue in trilysine. This suggests that the ionized ammonium group, which lysine and ornithine side chains have in common, is the main determinant of their conformational propensities at the central position in the respective tripeptides. The results of a DFT-based geometry optimization confirm this notion. In principle, our results suggest that lysine-rich segments in unfolded/disordered proteins and peptides can switch between different types of local order, i.e., an extended pPII(d)-like conformation and transient turns. However, for longer polylysine segments nonlocal interactions between side chains might impede the formation of turns, thus enabling the formation of pPII(d)-helix segments.

Triaspartate: a model system for conformationally flexible DDD motifs in proteins.

Understanding the interactions that govern turn formation in the unfolded state of proteins is necessary for a complete picture of the role that these turns play in both normal protein folding and functionally relevant yet disordered linear motifs. It is still unclear, however, whether short peptides can adopt stable turn structures in aqueous environments in the absence of any nonlocal interactions. To explore the effect that nearest-neighbor interactions and the local peptide environment have on the turn-forming capability of individual amino acid residues in short peptides, we combined vibrational (IR, Raman, and VCD), UV-CD, and (1)H NMR spectroscopies in order to probe the conformational ensemble of the central aspartic acid residue of the triaspartate peptide (DDD). The study was motivated by the recently discovered turn propensities of aspartic acid in GDG (Hagarman; et al. Chem.-Eur. J. 2011, 17, 6789). We investigated the DDD peptide under both acidic and neutral conditions in order to elucidate the effect that side-chain protonation has on the conformational propensity of the central aspartic acid residue. Amide I' profiles were analyzed in terms of two-dimensional Gaussian distributions representing conformational subdistributions in Ramachandran space. Interestingly, our results show that while the protonated form of the DDD peptide samples various turn-like conformations similar to GDG, deprotonation of the peptide eliminates this propensity for turns, causing the fully ionized peptide to exclusively sample pPII and ß-strand-like structures. To further explore the factors stabilizing these more extended conformations in fully ionized DDD, we analyzed the temperature dependence of both the UV-CD spectrum and the (3)J(H(N),H(α)) coupling constants of the two amide protons (N- and C-terminal) in terms of a simple two-state (pPII-ß) thermodynamic model. Thus, we were able to obtain the enthalpic and entropic differences between the pPII and ß-strand conformations of the central and C-terminal residue. For the central residue, we obtained ΔH(3) = -12.0 kJ/mol and ΔS(3) = -73.8 J/mol·K, resulting in a much larger room-temperature Gibbs free energy of 10.0 kJ/mol, which effectively locks the C-terminal in a ß-like conformation. A comparison of the temperature dependence of the chemical shifts reveals that there is indeed some type of protection of the amide protons from solvent in ionized DDD. This finding and several other lines of evidence suggest that both conformations of ionized DDD are stabilized by hydrogen bonding between the carboxylate groups of the central and C-terminal residue and the respective amide protons. These hydrogen bonds can be expected to be eliminated by side-chain protonation and substituted by hydrogen bonds between the N-terminal amide proton and the C-terminal carbonyl group as well as between the central aspartate side chain and the N-terminal amide proton. Hence, our results are indicative of a pH-induced switch in hydrogen-bonding patterns of aspartic acid motifs.

Conformational changes of trialanine induced by direct interactions between alanine residues and alcohols in binary mixtures of water with glycerol and ethanol.

Despite the increasing relevance of characterizing local conformational distributions in the unfolded state, an unambiguous description of the role that solvation and the addition of certain cosolvents play in altering this ensemble has yet to emerge. Alcohol cosolvents, and specifically glycerol, are known to act as protein stabilizers. The underlying mechanism of this effect is, however, still debated. Short alanine-based peptides provide a suitable model system for exploring the influence of cosolvents on backbone conformations, as ample experimental evidence now indicates that alanine does not exhibit a true statistical coil behavior but rather shows strong preference for sampling the polyproline II (PPII) region of the Ramachadran map when solvated in water. To explore the effect glycerol and ethanol cosolvents have on the conformational distribution of trialanine, we combined UV-CD and H NMR spectroscopies. The temperature dependence of the conformationally sensitive maximum dichroism (Δε) and (3)J(H(α)H(N)) coupling constants of two amide protons (N- and C-terminal) was subjected to a global thermodynamic analysis based on simple two-state PPIIâ†”ß models. Interestingly, our results show that even small admixtures of alcohol (5% v/v) considerably change the spectral parameters, Δε(PPII) and Δε(ß), as well as the enthalpic and entropic differences between the two states. For the central residue of trialanine in 5% glycerol, we obtained a gain in enthalpy favoring PPII of ΔΔH(n) = -4.80 kJ/mol and a compensating increase in entropy favoring the ß-strand of ΔΔS(n) = -13.53[J/mol K]. This causes increases in -ΔG and slight increases in PPII content. Further addition of alcohol, however, reverses the trend in that it causes a destabilization of the hydration shell and a shift toward ß-strand conformations. The combined manifold of ΔH and ΔS values obtained for the investigated binary mixtures and the pure aqueous solvent exhibits an excellent linear correlation, which reflects enthalpy-entropy compensation and a common transition temperature. The latter can be considered an indication of a weak binding between cosolvent and peptide. A comparison of infrared and Raman spectra of trialanine in water and in water-alcohol mixtures indeed reveals a close proximity between aliphatic side chains of alanine residues and alcohol molecules even for 5% (v/v) alcohol-water mixtures. Hence, our results provide the first experimental evidence for direct interactions between, e.g., glycerol and peptides in aqueous solutions, in line with the result of recent calculations by Vagenende et al. (Biochemistry 2009, 48, 11084-11096) but at variance with preferential exclusion theories.