Alanine-based Peptides Research Articles

Self-assembly of FRV3FR peptide into supramolecular nanofibrils

Small peptides are commonly used monomers for the self-assembly of biologically relevant supramolecular nanomaterials. In this study, the heptameric sequence FRV3FR was found to self-assemble into nanofibrils in solution, as confirmed by transmission electron microscopy (TEM), leading to the subsequent formation of hydrogels. Circular dichroism (CD) analysis showed that the FRV3FR peptide adopts a secondary structure that closely resembles a β-turn type conformation. An alanine-based peptide termed FRA3FR was also studied to highlight the impact of the middle amino acid triad has in self-assembly. Even though the FRA3FR showed a similar CD signal, it failed to provide any significant nanostructure or hydrogel formation, demonstrating the ability valine residues over alanine in promoting self-assembly. This work presents preliminary studies on a novel peptide sequence, laying the groundwork for its potential development into a functional peptide-based nanomaterial.

Effects of Proline on Internal Friction in Simulated Folding Dynamics of Several Alanine-Based α-Helical Peptides.

We have studied in silico the effect of proline, a model cosolvent, on local and global friction coefficients in (un)folding of several typical alanine-based α-helical peptides. Local friction is related to dwell times of a single, ensemble-averaged hydrogen bond (HB) within each peptide. Global friction is related to energy dissipated in a series of configurational changes of each peptide experienced by increasing the number of HBs during folding. Both of these approaches are important in relation to future atomic force microscopic-based measurements of internal friction via force-clamp single-molecule force spectroscopy. Molecular dynamics (MD) simulations for six peptides, namely, ALA5, ALA8, ALA15, ALA21, (AAQAA)3, and H2N-GN(AAQAA)2G-COONH2, have been conducted at 2 and 5 M proline solutions in water. Using previously obtained MD data for these peptides in pure water as well as upgraded theoretical models, we obtained variations of local and global internal friction coefficients as a function of solution viscosity. The results showed the substantial role of proline in stabilizing the folded state and slowing the overall folding dynamics. Consequently, larger friction coefficients were obtained at larger viscosities. The local and global internal friction, i.e., respective, friction coefficients approximated to zero viscosity, was also obtained. The evolution of friction coefficients with viscosity was weakly dependent on the number of concurrent folding pathways but was rather dominated by a stabilizing effect of proline on the folded states. Obtained values of local and global internal friction showed qualitatively similar results and a clear dependency on the structure of the studied peptide.

Length Dependent Folding Kinetics of Alanine-Based Helical Peptides from Optimal Dimensionality Reduction.

We present a computer simulation study of helix folding in alanine homopeptides (ALA)n of length n = 5, 8, 15, and 21 residues. Based on multi-microsecond molecular dynamics simulations at room temperature, we found helix populations and relaxation times increasing from about 6% and ~2 ns for ALA5 to about 60% and ~500 ns for ALA21, and folding free energies decreasing linearly with the increasing number of residues. The helix folding was analyzed with the Optimal Dimensionality Reduction method, yielding coarse-grained kinetic models that provided a detailed representation of the folding process. The shorter peptides, ALA5 and ALA8, tended to convert directly from coil to helix, while ALA15 and ALA21 traveled through several intermediates. Coarse-grained aggregate states representing the helix, coil, and intermediates were heterogeneous, encompassing multiple peptide conformations. The folding involved multiple pathways and interesting intermediate states were present on the folding paths, with partially formed helices, turns, and compact coils. Statistically, helix initiation was favored at both termini, and the helix was most stable in the central region. Importantly, we found the presence of underlying universal local dynamics in helical peptides with correlated transitions for neighboring hydrogen bonds. Overall, the structural and dynamical parameters extracted from the trajectories are in good agreement with experimental observables, providing microscopic insights into the complex helix folding kinetics.

Exposing the Nucleation Site of Alpha Helix Folding: A Joint Experimental and Simulation Study

α-helices being the most common secondary structures found ubiquitously in proteins, have been studied extensively for elucidating its folding dynamics and mechanism. In addition, the interest in studying and arriving at a mechanistic understanding of the α-helix formation is motivated by the effort to design and develop individually folded, stable α-helices with novel biological functions and therapeutic utilities. The kinetics of this helix folding has been fundamentally described by a series of discrete steps where the nucleation event is the slowest and occurs first, followed by a faster elongation process. Even for a homo-polypeptide, this α-helical structure is inherently asymmetric, due to the specific alignment of the helical H-bonds between i and i+4 amino acid residues in the sequence. Thus, the key question remains whether the inherent asymmetry induces a directionality in the folding pathway; i.e. the productive nucleation event occurs randomly or at a specific location within the peptide sequence. To address this question, we have utilized crosslinking strategy to stabilize α-helical conformation in short alanine-based peptides to introduce specifically localized nucleation sites along the peptide chain. Laser induced temperature-jump (T-jump) infrared (IR) spectroscopy and molecular dynamics (MD) simulations have been then used on these pre-nucleated peptides to show that indeed the folding occurs faster when the helix is nucleated at the N-terminus rather than at the C-terminus of the peptides, giving a directionality to helix formation.

N-terminal diproline and charge group effects on the stabilization of helical conformation in alanine-based short peptides: CD studies with water and methanol as solvent.

Protein folding problem remains a formidable challenge as main chain, side chain and solvent interactions remain entangled and have been difficult to resolve. Alanine-based short peptides are promising models to dissect protein folding initiation and propagation structurally as well as energetically. The effect of N-terminal diproline and charged side chains is assessed on the stabilization of helical conformation in alanine-based short peptides using circular dichroism (CD) with water and methanol as solvent. A1 (Ac-Pro-Pro-Ala-Lys-Ala-Lys-Ala-Lys-Ala-NH2 ) is designed to assess the effect of N-terminal homochiral diproline and lysine side chains to induce helical conformation. A2 (Ac-Pro-Pro-Glu-Glu-Ala-Ala-Lys-Lys-Ala-NH2 ) and A3 (Ac-dPro-Pro-Glu-Glu-Ala-Ala-Lys-Lys-Ala-NH2 ) with N-terminal homochiral and heterochiral diproline, respectively, are designed to assess the effect of Glu...Lys (i, i+4) salt bridge interactions on the stabilization of helical conformation. The CD spectra of A1, A2 and A3 in water manifest different amplitudes of the observed polyproline II (PPII) signals, which indicate different conformational distributions of the polypeptide structure. The strong effect of solvent substitution from water to methanol is observed for the peptides, and CD spectra in methanol evidence A2 and A3 as helical folds. Temperature-dependent CD spectra of A1 and A2 in water depict an isodichroic point reflecting coexistence of two conformations, PPII and β-strand conformation, which is consistent with the previous studies. The results illuminate the effect of N-terminal diproline and charged side chains in dictating the preferences for extended-β, semi-extended PPII and helical conformation in alanine-based short peptides. The results of the present study will enhance our understanding on stabilization of helical conformation in short peptides and hence aid in the design of novel peptides with helical structures. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Monte Carlo Simulations of the Effect of a Turn Sequence in an Alanine-Based Peptide

Alanine-based peptides are known to form single alpha helical chains. Furthermore, PSDP sequences form turns in the two-helix bundle needle proteins from type III secretion systems. To determine if the PSDP sequence would bias an alanine-based peptide toward a two helix bundle, we have carried out Monte Carlo simulations of an alanine-based peptide with and without a PSDP sequence in the middle of the peptide. Because needle proteins are 40 to 45 residues in length, we used 42 residue peptides in our simulations. The alanine-based peptide consisted of a (AAAAAK)7 sequence which we call AK42, whereas the AK42_PSDP peptide inserted a PSDP sequence centrally by replacing the fourth repeat of (AAAAAK)7 with APSDPK. We carried out Replica Exchange Monte Carlo simulations on the two peptides using the CAMPARI simulation package. A total of 16 temperatures ranging from 280 K to 460 K were used to monitor the globule to coil transition for our two peptide models. Our data show a stabilization of the globule form of AK42_PSDP to higher temperatures when monitoring the radius of gyration (Rg) compared to that of AK42. We will also report on the position of chain reversals using clustering algorithms which allow us to see prominent structures at each temperature and thus the influence of the PSDP sequence on the narrowness of the conformational distribution of the alanine-based peptide.

Microscopic nucleation and propagation rates of an alanine-based α-helix.

An infrared temperature-jump (T-jump) study by Huang et al. (Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 2788-2793) showed that the conformational relaxation kinetics of an alanine-based α-helical peptide depend not only on the final temperature (Tf) but also on the initial temperature (Ti) when Tf is fixed. Their finding indicates that the folding free energy landscape of this peptide is non-two-state like, allowing for the population of conformational ensembles with different helical lengths and relaxation times in the temperature range of the experiment. Because α-helix folding involves two fundamental events, nucleation and propagation, the results of Huang et al. thus present a unique opportunity to determine their rate constants - a long-sought goal in the study of the helix-coil transition dynamics. Herein, we capitalize on this notion and develop a coarse-grained kinetic model to globally fit the thermal unfolding curve and T-jump kinetic traces of this peptide. Using this strategy, we are able to explicitly determine the microscopic rate constants of the kinetic steps encountered in the nucleation and propagation processes. Our results reveal that the time taken to form one α-helical turn (i.e., an α-helical segment with one helical hydrogen bond) is about 315 ns, whereas the time taken to elongate this nucleus by one residue (or backbone unit) is 5.9 ns, depending on the position of the residue.

Influence of Glu/Arg, Asp/Arg, and Glu/Lys Salt Bridges on α-Helical Stability and Folding Kinetics

Using a combination of ultraviolet circular dichroism, temperature-jump transient-infrared spectroscopy, and molecular dynamics simulations, we investigate the effect of salt bridges between different types of charged amino-acid residue pairs on α-helix folding. We determine the stability and the folding and unfolding rates of 12 alanine-based α-helical peptides, each of which has a nearly identical composition containing three pairs of positively and negatively charged residues (either Glu−/Arg+, Asp−/Arg+, or Glu−/Lys+). Within each set of peptides, the distance and order of the oppositely charged residues in the peptide sequence differ, such that they have different capabilities of forming salt bridges. Our results indicate that stabilizing salt bridges (in which the interacting residues are spaced and ordered such that they favor helix formation) speed up α-helix formation by up to 50% and slow down the unfolding of the α-helix, whereas salt bridges with an unfavorable geometry have the opposite effect. Comparing the peptides with different types of charge pairs, we observe that salt bridges between side chains of Glu− and Arg+ are most favorable for the speed of folding, probably because of the larger conformational space of the salt-bridging Glu−/Arg+ rotamer pairs compared to Asp−/Arg+ and Glu−/Lys+. We speculate that the observed impact of salt bridges on the folding kinetics might explain why some proteins contain salt bridges that do not stabilize the final, folded conformation.

Using Kinetic Network Models To Probe Non-Native Salt-Bridge Effects on α-Helix Folding.

Salt-bridge interactions play an important role in stabilizing many protein structures, and have been shown to be designable features for protein design. In this work, we study the effects of non-native salt bridges on the folding of a soluble alanine-based peptide (Fs peptide) using extensive all-atom molecular dynamics simulations performed on the Folding@home distributed computing platform. Using Markov State Models, we show how non-native salt-bridges affect the folding kinetics of Fs peptide by perturbing specific conformational states. Furthermore, we present methods for the automatic detection and analysis of such states. These results provide insight into helix folding mechanisms and useful information to guide simulation-based computational protein design.

Insights into Unfolded Proteins from the Intrinsic ϕ/ψ Propensities of the AAXAA Host-Guest Series

Various host-guest peptide series are used by experimentalists as reference conformational states. One such use is as a baseline for random-coil NMR chemical shifts. Comparison to this random-coil baseline, through secondary chemical shifts, is used to infer protein secondary structure. The use of these random-coil data sets rests on the perception that the reference chemical shifts arise from states where there is little or no conformational bias. However, there is growing evidence that the conformational composition of natively and nonnatively unfolded proteins fail to approach anything that can be construed as random coil. Here, we use molecular dynamics simulations of an alanine-based host-guest peptide series (AAXAA) as a model of unfolded and denatured states to examine the intrinsic propensities of the amino acids. We produced ensembles that are in good agreement with the experimental NMR chemical shifts and confirm that the sampling of the 20 natural amino acids in this peptide series is be far from random. Preferences toward certain regions of conformational space were both present and dependent upon the environment when compared under conditions typically used to denature proteins, i.e., thermal and chemical denaturation. Moreover, the simulations allowed us to examine the conformational makeup of the underlying ensembles giving rise to the ensemble-averaged chemical shifts. We present these data as an intrinsic backbone propensity library that forms part of our Structural Library of Intrinsic Residue Propensities to inform model building, to aid in interpretation of experiment, and for structure prediction of natively and nonnatively unfolded states.

Gelation of Highly Cationic Alanine Based Peptide in Water in Absence of Charge Screening Anions

Peptide aggregation and self-assembly is of interest due to the possible link to many neurodegenerative diseases, as well as possible biotechnological applications. Surprisingly, the alanine-based peptide, (AAKA)4, (“AK-16”) has been previously shown to aggregate and form a hydrogel in the presence of a sufficiently high concentration of anions, although it may seem intuitive that the positive lysine residues would prevent aggregation. Here, we report the delayed self-assembly of pre-aggregated AK-16 into a hydrogel in the absence of neutralizing anions. Self assembly kinetics was probed by both IR and vibrational circular dichroism. At low concentrations (<15mg/ml), the peptide initially forms β-sheet rich structures, which decays over a 5 day period, as evidenced by a decrease in the intensity of the amide I’ band at 1616cm-1. This decay likely indicates the formation of more disordered structures or amorphous aggregates. At higher concentrations, the β-sheet content remains stable over a much longer time scale, and a stable hydrogel eventually forms. The intrinsic intensity of the amide I’ band significantly increases upon gelation, revealing it as a spectroscopic marker for gelation. We attribute this to an increasing hydration of the peptide backbone. Moreover, the structural decay into amorphous aggregates as well as hydrogelation lead to rather substantial spectroscopic changes in the 1350 −1500 cm-1 region of the IR spectrum. AFM images reveal a complex nanoweb structure of the gelated peptide which is typical of noncovalently crosslinked fibrils. This cationic system lacks complimentary charges, a common feature of peptide hydrogels, and offers promise in regards to biotechnological applications.

Action-FRET: probing the molecular conformation of mass-selected gas-phase peptides with Förster resonance energy transfer detected by acceptor-specific fragmentation.

The use of Förster resonance energy transfer (FRET) as a probe of the structure of biological molecules through fluorescence measurements in solution is well-attested. The transposition of this technique to the gas phase is appealing since it opens the perspective of combining the structural accuracy of FRET with the specificity and selectivity of mass spectrometry (MS). Here, we report FRET results on gas-phase polyalanine ions obtained by measuring FRET efficiency through specific photofragmentation rather than fluorescence. The structural sensitivity of the method was tested using commercially available chromophores (QSY 7 and carboxyrhodamine 575) grafted on a series of small, alanine-based peptides of differing sizes. The photofragmentation of these systems was investigated through action spectroscopy, and their conformations were probed using ion mobility spectrometry (IMS) and Monte Carlo minimization (MCM) simulations. We show that specific excitation of the donor chromophore results in the observation of fragments that are specific to the electronic excitation of the acceptor chromophore. This shows that energy transfer took place between the two chromophores and hence that the action-FRET technique can be used as a new and sensitive probe of the structure of gas-phase biomolecules, which opens perspectives as a new tool in structural biology.

Slow Folding–Unfolding Kinetics of an Octameric β-Peptide Bundle

β-Peptide foldamers offer attractive frameworks for examining the effect of backbone flexibility on the dynamics of protein folding. Herein, we study the folding-unfolding kinetics of a β-peptide, Acid-1Y,1 which folds in aqueous solution into an octameric bundle of peptides in a conformation known as the 14-helix. Acid-1Y is comprised exclusively of β-amino acids, which differ from α-amino acids by the addition of a single methylene into the backbone. We aim to understand how the additional degree of freedom and increased backbone flexibility in the β-amino acid affect folding dynamics and to measure folding rates of this octameric β-peptide. Previously, we found that the T-jump induced relaxation kinetics of a monomeric β-peptide that forms a monomeric 14-helix occurred on the nanosecond time scale2 and were noticeably slower than a similar alanine-based α-helical peptide.3 Additionally, in comparison to similar α-helices, the relaxation rates showed a weaker dependence on temperature. Here, we find that the T-jump induced relaxation kinetics of the octameric β-peptide occurs on an even slower time scale (minutes) and the unfolding relaxation rates show a large dependence on temperature. These differences indicate that folding energy landscapes of β-peptide secondary and quaternary structure are markedly distinct from one another and also from their α-helical counterparts.

Helix Formation by Alanine-Based Peptides in Pure Water and Electrolyte Solutions: Insights from Molecular Dynamics Simulations

Specific ion effects on oligopeptide conformations in solution are attracting strong research attention, because of their impact on the protein-folding problem and on several important biological-biotechnological applications. In this work, we have addressed specific effects of electrolytes on the tendency of oligopeptides toward formation and propagation of helical segments. We have used replica-exchange molecular dynamics (REMD) simulations to study the conformations of two short hydrophobic peptides [Ace-(AAQAA)3-Nme (AQ), and Ace-A8-Nme (A8)] in pure water and in aqueous solutions of sodium chloride (NaCl) and sodium iodide (NaI) with concentrations of 1 and 3 M. The average helicities of the AQ peptide have been analyzed to yield Lifson-Roig (LR) parameters for helix nucleation and helix propagation. The salt dependence of these parameters suggests that electrolytes tend to stabilize the helical conformations of short peptides by enhancing the helix nucleation parameter. The helical conformations of longer oligopeptides are destabilized in the presence of salts, however, because the helix propagation parameters are reduced by electrolytes. On top of this general trend, we observe a significant specific salt effect in these simulations. The hydrophobic iodide ion in NaI solutions has a high affinity for the peptide backbone, which reflects itself in an enhanced helix nucleation and a reduced helix propagation parameter with respect to pure water or NaCl solutions. The present work thus explains the computational evidence that electrolytes tend to stabilize the compact conformations of short peptides and destabilize them for longer peptides, and it also sheds additional light on the specific salt effects on compact peptide conformations.

The intrinsic conformational features of amino acids from a protein coil library and their applications in force field development

The local conformational (φ, ψ, χ) preferences of amino acid residues remain an active research area, which are important for the development of protein force fields. In this perspective article, we first summarize spectroscopic studies of alanine-based short peptides in aqueous solution. While most studies indicate a preference for the P(II) conformation in the unfolded state over α and β conformations, significant variations are also observed. A statistical analysis from various coil libraries of high-resolution protein structures is then summarized, which gives a more coherent view of the local conformational features. The φ, ψ, χ distributions of the 20 amino acids have been obtained from a protein coil library, considering both backbone and side-chain conformational preferences. The intrinsic side-chain χ(1) rotamer preference and χ(1)-dependent Ramachandran plot can be generally understood by combining the interaction of the side-chain Cγ/Oγ atom with two neighboring backbone peptide groups. Current all-atom force fields such as AMBER ff99sb-ILDN, ff03 and OPLS-AA/L do not reproduce these distributions well. A method has been developed by combining the φ, ψ plot of alanine with the influence of side-chain χ(1) rotamers to derive the local conformational features of various amino acids. It has been further applied to improve the OPLS-AA force field. The modified force field (OPLS-AA/C) reproduces experimental (3)J coupling constants for various short peptides quite well. It also better reproduces the temperature-dependence of the helix-coil transition for alanine-based peptides. The new force field can fold a series of peptides and proteins with various secondary structures to their experimental structures. MD simulations of several globular proteins using the improved force field give significantly less deviation (RMSD) to experimental structures. The results indicate that the local conformational features from coil libraries are valuable for the development of balanced protein force fields.

Impact of ion binding on poly-L-lysine (un)folding energy landscape and kinetics.

We utilize T-jump UV resonance Raman spectroscopy (UVRR) to study the impact of ion binding on the equilibrium energy landscape and on (un)folding kinetics of poly-L-lysine (PLL). We observe that the relaxation rates of the folded conformations (including π-helix (bulge), pure α-helix, and turns) of PLL are slower than those of short alanine-based peptides. The PLL pure α-helix folding time is similar to that of short alanine-based peptides. We for the first time have directly observed that turn conformations are α-helix and π-helix (bulge) unfolding intermediates. ClO(4)(-) binding to the Lys side chain -NH(3)(+) groups and the peptide backbone slows the α-helix unfolding rate compared to that in pure water, but little impacts the folding rate, resulting in an increased α-helix stability. ClO(4)(-) binding significantly increases the PLL unfolding activation barrier but little impacts the folding barrier. Thus, the PLL folding coordinate(s) differs from the unfolding coordinate(s). The-π helix (bulge) unfolding and folding coordinates do not directly go through the α-helix energy well. Our results clearly demonstrate that PLL (un)folding is not a two-state process.

Residue-Specific α-Helix Propensities from Molecular Simulation

Formation of α-helices is a fundamental process in protein folding and assembly. By studying helix formation in molecular simulations of a series of alanine-based peptides, we obtain the temperature-dependent α-helix propensities of all 20 naturally occurring residues with two recent additive force fields, Amber ff03w and Amber ff99SB∗. Encouragingly, we find that the overall helix propensity of many residues is captured well by both energy functions, with Amber ff99SB∗ being more accurate. Nonetheless, there are some residues that deviate considerably from experiment, which can be attributed to two aspects of the energy function: i), variations of the charge model used to determine the atomic partial charges, with residues whose backbone charges differ most from alanine tending to have the largest error; ii), side-chain torsion potentials, as illustrated by the effect of modifications to the torsion angles of I, L, D, N. We find that constrained refitting of residue charges for charged residues in Amber ff99SB∗ significantly improves their helix propensity. The resulting parameters should more faithfully reproduce helix propensities in simulations of protein folding and disordered proteins.

Like-charged residues at the ends of oligoalanine sequences might induce a chain reversal.

We have examined the effect of like-charged residues on the conformation of an oligoalanine sequence. This was facilitated by circular dichroism (CD) and NMR spectroscopic and differential scanning calorimetric (DSC) measurements, and molecular dynamics calculations of the following three alanine-based peptides: Ac-K-(A)(5) -K-NH(2) (KAK5), Ac-K-(A)(4) -K-NH(2) (KAK4), Ac-K-(A)(3) -K-NH(2) (KAK3), where A and K denote alanine and lysine residues, respectively. Our earlier studies suggested that the presence of like-charged residues at the end of a short polypeptide chain composed of nonpolar residues can induce a chain reversal. For all three peptides, canonical molecular dynamics simulations with NMR-derived restraints demonstrate the presence of ensembles of structures with a tendency to form a chain reversal. The KAK3 peptide exhibits a bent shape with its ends close to each other, while KAK4 and KAK5 are more extended. In the KAK5 peptide, the lysine residues do not have any influence on each other and are very mobile. Nevertheless, the tendency to form a more or less pronounced chain reversal is observed and it seems to be stable in all three peptides. This chain reversal seems to be caused by screening of the nonpolar core from the solvent by the hydrated charged residues.

Host–Guest Chemistry in the Gas Phase: Complex Formation with 18-Crown-6 Enhances Helicity of Alanine-Based Peptides

The gas-phase helix propensities of alanine-based polypeptides are studied with different locations of a Lys residue and host-guest interactions with 18-Crown-6 (18C6). A series of model peptides Ac-Ala(9-n)-LysH(+)-Ala(n) (n = 0, 1, 3, 5, 7, and 9) is examined alone and with 18C6 using traveling wave ion mobility mass spectrometry combined with molecular dynamics (MD) simulations. The gas-phase helices are observed from the peptides whose Lys residue is located close to the C-terminus so that the Lys exerts its capping effect on the C-terminal carbonyl groups. The peptides, which interact with 18C6 in the gas phase, show enhanced helical propensities. The enhanced helicity of the peptide in the complex is attributed by isolation of the Lys butylammonium group from the helix backbone and the interaction of methylene groups of 18C6, which possess localized positive partial charges, with C-terminal carbonyl groups serving as a cap to stabilize the helix.

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 PPII↔β models. Interestingly, our results show that even small admixtures of alcohol (5% v/v) considerably change the spectral parameters, Δε(PPII) and Δε(β), 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 β-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 β-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.