Short Alanine Peptides Research Articles

Dipole Cooperativity and Polarization Frustration Determine the Secondary Structure Distribution of Short Alanine Peptides in Water.

The physical driving forces for secondary structure preferences of hydrated alanine peptide are investigated with B3LYP-D3(BJ) and the adaptive force matching (AFM) method. The AFM fit to the DFT surface, ALA2022, provides excellent agreement with the nuclear magnetic resonance scalar coupling constants from experiments. In turn, the model is used to gain insight into the physical driving forces behind secondary structure preferences of hydrated peptides. DFT calculations with and without the Conductor-like Screening Model (COSMO) show that the α helix is stabilized by solvent polarization due to dipole cooperativity. The two adjacent amide groups in β strand form a near-planar trapezoid that is not much larger than the size of water molecules. When the finite size of a water molecule is considered, the stabilization from solvent polarization for such a trapezoid is frustrated. Water molecules cannot find orientations to properly stabilize all four polar regions close to each other with such an awkward arrangement. This leads to quite substantial reduction in polarization stabilization. Although the polyproline II (PP-II) conformation is very similar to the β strand, the small twist in the backbone angles allowed much improved polarization stabilization. The improved polarization, when combined with favorable intrapeptide interactions, leads to the PP-II to be lowest in free energy. Other factors, such as the entropic TΔS and the ϕ, ψ coupling terms, are also studied but are found to play only a minor role. The insight shown in this work helps to better understand the structure of globular and intrinsic disordered proteins and facilitate future force field development.

Depolarizing Effects in Hydrogen Bond Energy in 310-Helices Revealed by Quantum Chemical Analysis.

Hydrogen-bond (H-bond) energies in 310-helices of short alanine peptides were systematically examined by precise DFT calculations with the negative fragmentation approach (NFA), a modified method based on the molecular tailoring approach. The contribution of each H-bond was evaluated in detail from the 310-helical conformation of total energies (whole helical model, WH3-10 model), and the results were compared with the property of H-bond in α-helix from our previous study. The H-bond energies of the WH3-10 model exhibited tendencies different from those exhibited by the α-helix in that they depended on the helical position of the relevant H-bond pair. H-bond pairs adjacent to the terminal H-bond pairs were observed to be strongly destabilized. The analysis of electronic structures indicated that structural characteristics cause the destabilization of the H-bond in 310-helices. We also found that the longer the helix length, the more stable the H-bond in the terminal pairs of the WH3-10 model, suggesting the action of H-bond cooperativity.

Hydrogen bond donors and acceptors are generally depolarized in α-helices as revealed by a molecular tailoring approach.

Hydrogen‐bond (H‐bond) interaction energies in α‐helices of short alanine peptides were systematically examined by precise density functional theory calculations, followed by a molecular tailoring approach. The contribution of each H‐bond interaction in α‐helices was estimated in detail from the entire conformation energies, and the results were compared with those in the minimal H‐bond models, in which only H‐bond donors and acceptors exist with the capping methyl groups. The former interaction energies were always significantly weaker than the latter energies, when the same geometries of the H‐bond donors and acceptors were applied. The chemical origin of this phenomenon was investigated by analyzing the differences among the electronic structures of the local peptide backbones of the α‐helices and those of the minimal H‐bond models. Consequently, we found that the reduced H‐bond energy originated from the depolarizations of both the H‐bond donor and acceptor groups, due to the repulsive interactions with the neighboring polar peptide groups in the α‐helix backbone. The classical force fields provide similar H‐bond energies to those in the minimal H‐bond models, which ignore the current depolarization effect, and thus they overestimate the actual H‐bond energies in α‐helices. © 2019 The Authors. Journal of Computational Chemistry published by Wiley Periodicals, Inc.

Peptide Conformation Analysis Using an Integrated Bayesian Approach.

Unlike native proteins that are amenable to structural analysis at atomic resolution, unfolded proteins occupy a manifold of dynamically interconverting structures. Defining the conformations of unfolded proteins is of significant interest and importance, for folding studies and for understanding the properties of intrinsically disordered proteins. Short chain protein fragments, i.e., oligopeptides, provide an excellent test-bed in efforts to define the conformational ensemble of unfolded chains. Oligomers of alanine in particular have been extensively studied as minimalist models of the intrinsic conformational preferences of the peptide backbone. Even short alanine peptides occupy an ensemble of substates that are distinguished by small free energy differences, so that the problem of quantifying the conformational preferences of the backbone remains a fundamental challenge in protein biophysics. Here, we demonstrate an integrated computational-experimental-Bayesian approach to quantify the conformational ensembles of the model trialanine peptide in water. In this approach, peptide conformational substates are first determined objectively by clustering molecular dynamics snapshots based on both structural and dynamic information. Next, a set of spectroscopic data for each conformational substate is computed. Finally, a Bayesian statistical analysis of both experimentally measured spectroscopic data and computational results is carried out to provide a current best estimate of the substate population ensemble together with corresponding confidence intervals. This distribution of substates can be further systematically refined with additional high-quality experimental data and more accurate computational modeling. Using an experimental data set of NMR coupling constants, we have also applied this approach to characterize the conformation ensemble of trivaline in water.

Contribution of the empirical dispersion correction on the conformation of short alanine peptides obtained by gas-phase QM calculations.

In this work we analyze the effect of the inclusion of an empirical dispersion term to standard DFT (DFT-D) in the prediction of the conformational energy of the alanine dipeptide (Ala2) and in assessing the relative stabilities of short polyala-nine peptides in helical conformations, i.e., α and 310 helices, from Ala4 to Ala16. The Ala2 conformational energies obtained with the dispersion-corrected GGA functional B97-D are compared to previously published high level MP2 data. Meanwhile, the B97-D performance on larger polyalanine peptides is compared to MP2, B3LYP and RHF calculations obtained at a lower level of theory. Our results show that electron correlation affects the conformational energies of short peptides with a weight that increases with the peptide length. Indeed, while the contribution of vdW forces is significant for larger peptides, in the case of Ala2 it is negligible when compared to solvent effects. Even for short peptides, the inclusion of an empirical dispersion term greatly improves accuracy of DFT methods, providing results that correlate very well with the MP2 reference at no additional computational cost.

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.

Antifreeze glycoprotein agents: structural requirements for activity

Antifreeze glycoproteins (AFGPs) are considered to be the most efficient means to reduce ice damage to cell tissues since they are able to inhibit growth and crystallization of ice. The key element of antifreeze proteins is to act in a non-colligative manner which allows them to function at concentrations 300-500 times lowers than other dissolved solutes. During the past decade, AFGPs have demonstrated tremendous potential for many pharmaceutical and food applications. Presently, the only route to obtain AFGPs involves the time consuming and expensive process of isolation and purification from deep-sea polar fishes. Unfortunately, it is not amenable to mass production and commercial applications. The lack of understanding of the mechanism through which the AFGPs inhibit ice growth has also hampered the realization of industrial and biotechnological applications. Here we report the structural motifs that are essential for antifreeze activity of AFGPs, and propose a unified mechanism based on both recent studies of short alanine peptides and structure activity relationship of synthesized AFGPs.

Spin Relaxation Enhancement Confirms Dominance of Extended Conformations in Short Alanine Peptides

Local order goes with random coil: An ensemble of peptide structures is generated with over 90 % extended conformation for Ala and Pro. The population-averaged long-range distance from the N-terminal spin-label residue (see picture, red dot) to the C-terminal amide proton of Ala (blue dots) is fully consistent with the results from NMR spin relaxation measurements.

Spin Relaxation Enhancement Confirms Dominance of Extended Conformations in Short Alanine Peptides

Lokale Ordnung und Zufallsknäuel: Ein Ensemble von Peptidstrukturen mit über 90 % gestreckter Konformation für Ala und Pro wurde erzeugt. Der mittlere Abstand einer N-terminalen Spinmarkierung (roter Punkt im Bild) zu den C-terminalen Amidprotonen von Ala (blaue Punkte) bestätigte die Ergebnisse von NMR-Spinrelaxationsmessungen.

Conformations of Alanine-Based Peptides in Water Probed by FTIR, Raman, Vibrational Circular Dichroism, Electronic Circular Dichroism, and NMR Spectroscopy

We have used a combination of FTIR, VCD, ECD, Raman, and NMR spectroscopies to probe the solution conformations sampled by H-(AAKA)-OH by utilizing an excitonic coupling model and constraints imposed by the 3JCalphaHNH coupling constants of the central residues to simulate the amide I' profile of the IR, isotropic Raman, anisotropic Raman, and VCD spectra in terms of a mixture of three conformations, i.e., polyproline II, beta-strand and right-handed helical. The representative coordinates of the three conformations were obtained from published coil libraries. Alanine was found to exhibit PPII fractions of 0.60 or greater, mixed with smaller fractions of helices and beta-strand conformations. Lysine showed no clear conformational propensity in that it samples polyproline II, beta-strand, and helical conformations with comparable probability. This is at variance with results obtained earlier for ionized polylysine, which suggest a high polyproline II propensity. We reanalyzed previously investigated tetra- and trialanine by combining published vibrational spectroscopy data with 3JCalphaHNH coupling constants and obtained again blends dominated by PPII with smaller admixtures of beta-strand and right-handed helical conformations. The polyproline II propensity of alanine was found to be higher in tetraalanine than in trialanine. For all peptides investigated, our results rule out a substantial population of turn-like conformations. Our results are in excellent agreement with MD simulations on short alanine peptides by Gnanakaran and Garcia [(2003) J. Phys. Chem. B 107, 12555-12557] but at variance with multiple MD simulations particularly for the alanine dipeptide.

Comparison of multiple Amber force fields and development of improved protein backbone parameters

The ff94 force field that is commonly associated with the Amber simulation package is one of the most widely used parameter sets for biomolecular simulation. After a decade of extensive use and testing, limitations in this force field, such as over-stabilization of alpha-helices, were reported by us and other researchers. This led to a number of attempts to improve these parameters, resulting in a variety of "Amber" force fields and significant difficulty in determining which should be used for a particular application. We show that several of these continue to suffer from inadequate balance between different secondary structure elements. In addition, the approach used in most of these studies neglected to account for the existence in Amber of two sets of backbone phi/psi dihedral terms. This led to parameter sets that provide unreasonable conformational preferences for glycine. We report here an effort to improve the phi/psi dihedral terms in the ff99 energy function. Dihedral term parameters are based on fitting the energies of multiple conformations of glycine and alanine tetrapeptides from high level ab initio quantum mechanical calculations. The new parameters for backbone dihedrals replace those in the existing ff99 force field. This parameter set, which we denote ff99SB, achieves a better balance of secondary structure elements as judged by improved distribution of backbone dihedrals for glycine and alanine with respect to PDB survey data. It also accomplishes improved agreement with published experimental data for conformational preferences of short alanine peptides and better accord with experimental NMR relaxation data of test protein systems.

Strike a balance: Optimization of backbone torsion parameters of AMBER polarizable force field for simulations of proteins and peptides

Based on the AMBER polarizable model (ff02), we have re-optimized the parameters related to the main-chain (Phi, Psi) torsion angles by fitting to the Boltzmann-weighted average quantum mechanical (QM) energies of the important regions (i.e., beta, P(II), alpha(R), and alpha(L) regions). Following the naming convention of the AMBER force field series, this release will be called ff02pol.rl The force field has been assessed both by energetic comparison against the QM data and by the replica exchange molecular dynamics simulations of short alanine peptides in water. For Ace-Ala-Nme, the simulated populations in the beta, P(II) and alpha(R) regions were approximately 30, 43, and 26%, respectively. For Ace-(Ala)(7)-Nme, the populations in these three regions were approximately 24, 49, and 26%. Both were in qualitative agreement with the NMR and CD experimental conclusions. In comparison with the previous force field, ff02pol.rl demonstrated good balance among these three important regions. The optimized torsion parameters, together with those in ff02, allow us to carry out simulations on proteins and peptides with the consideration of polarization.

The polyproline II conformation in short alanine peptides is noncooperative.

The finding that short alanine peptides possess a high fraction of polyproline II (PII) structure (Phi=-75 degrees, Psi=+145 degrees ) at low temperature has broad implications for unfolded states of proteins. An important question concerns whether or not this structure is locally determined or cooperative. We have monitored the conformation of alanine in a series of model peptides AcGGAnGGNH2 (n=1-3) over a temperature range from -10 degrees C to +80 degrees C. Use of 15N-labeled alanine substitutions makes it possible to measure 3JalphaN coupling constants accurately over the full temperature range. Based on a 1D next-neighbor model, the cooperative parameter sigma of PII nucleation is evaluated from the coupling constant data. The finding that sigma is close to unity (1 +/- 0.2) indicates a noncooperative role for alanine in PII structure formation, consistent with statistical surveys of the Protein Data Bank that suggest that most PII structure occurs in isolated residues. Lack of cooperativity in these models implies that hydration effects that influence PII conformation in water are highly localized. Using a nuclear Overhauser effect ratio strategy to define the alanine Psi angle, we estimate that, at 40 degrees C, the time-averaged alanine conformation (Phi=-80 degrees, Psi=+170 degrees ) deviates from canonical PII structure, indicating that PII melts at high temperature. Thus, the high-temperature state of short alanine peptides seems to be an unfolded ensemble with higher distribution in the extended beta structure basin, but not a coil.

Water molecule adsorption on short alanine peptides: how short is the shortest gas-phase alanine-based helix?

Water adsorption measurements have been performed under equilibrium conditions for unsolvated Ac-A(n)K+H(+) and Ac-KA(n)+H(+) peptides with n = 4 - 10. Previous work on larger alanine peptides has shown that two dominant conformations (helices and globules) are present for these peptides and that water adsorbs much more strongly to the globules than to the helices. All the Ac-KA(n)+H(+) peptides studied here (which are expected to be globular) adsorb water strongly, and so do the Ac-A(n)K+H(+) peptides with n < 8. However, for Ac-A(n)K+H(+) with n = 8-10 there is a substantial drop in the propensity to adsorb water. This result suggests that Ac-A(8)K+H(+) is the smallest Ac-A(n)K+H(+) peptide to have a significant helical content in the gas phase. Water adsorption measurements for Ac-V(n)K+H(+) and Ac-L(n)K+H(+) with n = 5-10 suggest that the helix emerges at n = 8 for these peptides as well.

Validation of an All-Atom Protein Force Field: From Dipeptides to Larger Peptides

New experimental techniques are capable of determining the relative population of conformations adopted by short alanine peptides in water. Most of the existing all-atom force fields used to model proteins fail to reproduce the relative population of the most relevant conformations of peptides. The calculated relative population of conformations varies significantly depending on the force field chosen, thus urging the need to check the validity and consistency of force fields over a range of peptide lengths. Here, we show how the applicability of a modified version of AMBER force field (A94/MOD) can extend from short to large peptides. It is also capable of reproducing the expected shift in conformational preference with increasing peptide length and temperature. Importantly, the consistency of the force field is judged by direct comparison to experiments rather than to the relative energies of conformations obtained from ab initio calculations. Importantly, this study illustrates that many aspects of protein force fields are already well refined and may only require minor refinements to accurately reproduce experimental observations over a range of systems.

Polyproline II structure in a sequence of seven alanine residues.

A sequence of seven alanine residues-too short to form an alpha-helix and whose side chains do not interact with each other-is a particularly simple model for testing the common description of denatured proteins as structureless random coils. The (3)J(HN alpha) coupling constants of individual alanine residues have been measured from 2 to 56 degrees C by using isotopically labeled samples. The results display a thermal transition between different backbone conformations, which is confirmed by CD spectra. The NMR results suggest that polyproline II is the dominant conformation at 2 degrees C and the content of beta strand is increased by approximately 10% at 55 degrees C relative to that at 2 degrees C. The polyproline II conformation is consistent with recent studies of short alanine peptides, including structure prediction by ab initio quantum mechanics and solution structures for both a blocked alanine dipeptide and an alanine tripeptide. CD and other optical spectroscopies have found structure in longer "random coil" peptides and have implicated polyproline II, which is a major backbone conformation in residues within loop regions of protein structures. Our result suggests that the backbone conformational entropy in alanine peptides is considerably smaller than estimated by the random coil model. New thermodynamic data confirm this suggestion: the entropy loss on alanine helix formation is only 2.2 entropy units per residue.

Comparison of conformational behavior of the short alanine peptides and their boron analogues

Conformational behavior of short terminally blocked alanine peptides up to three residues has been analyzed by means of a new computer program Vader. The program is based on Single-Coordinate-Driving method and it employs quantum chemistry energy calculation. The semiempirical AM1 method has been used in this study. Conformational behavior of alanine peptides has been compared with their boron analogues. It has been found that: 1. Semiempirical AM1 method creates an artifact in energy calculation of boron analogues for geometries where the dihedral angles are around zero value. In such a case, ab initio methods have to be employed. 2. Boron analogues appear conformationally more flexible. They exhibit much higher number of energy minima along the PES. It has also been concluded that the program Vader is able to give quite a good estimation of stationary points. All stationary points found by Vader for monomers and those stationary points with relative energy less that 2.0 kcal/mol found for the dimers were subjected to frequency analysis of the Hessian matrix. In all cases, they were confirmed as stationary points of the same category as predicted by Vader.

Short alanine-based peptides may form 3(10)-helices and not alpha-helices in aqueous solution.

Short alanine peptides, containing 16 or 17 residues, appear to form alpha-helices in aqueous solution. But the main spectroscopic analyses used on helical peptides (circular dichroism and nuclear magnetic resonance) cannot distinguish between an alpha-helix (in which the ith residue is hydrogen-bonded to residue i + 4; ref. 9) and the next most common peptide helix, the 3(10)-helix10 (i-->i + 3 hydrogen-bonding). To address this problem we have designed single and doubly spin-labelled analogues of alanine-based peptides in which the nitroxide spin label forms an unbranched side chain extending from the sulphur atom of a cysteine residue. Here we report the circular dichroism, Fourier-transform infrared and electron-spin resonance spectra of these peptides under helix-forming conditions. The infrared absorbance gives an amide I' band with a frequency that is substantially different from that observed for alpha-helices. The electron-spin resonance spectra of doubly labelled helices show that the ranking of distances between side chains, around a single turn (residues 4-8), is inconsistent with an alpha-helical structure. Our experiments suggest that the more likely peptide geometry is a 3(10)-helix.