Conformational flexibility and loss of structural rigidity for a model hexapeptide,GRGDTP: 1H‐NMR and molecular dynamics studies

The NMR and molecular dynamics methods are used to study the conformations of a hexapeptide, GRGDTP, which has been shown to be accessible to various types of cell‐adhesion based cellular behaviors such as cell‐to‐matrix interactions, cell differentiation, immunogenicity development, gene expression, angiogenesis, metastasis, sex determination and gamete fusion. ¹H‐NMR results indicate the existence of weak 5→2 hydrogen bonded β‐turn type‐III. Molecular simulation studies using a mixed protocol of distance geometry, constrained minimization, restrained molecular dynamics followed by energy minimization resulted additional conformations that include about 64% of population of inverse γ‐turn (HB, 3→1) and about 35% population of γ‐turn (HB, 4→2). The inter‐proton distances observed in γ‐and inverse γ‐turns are also consistent with the NMR constraints. The variable internal hydrogen bonding due to γ‐turns initiated at Gly 1 and Arg 2 , and its tendency to inter‐convert between γ‐and inverse γ‐turn conformations imply that the peptide is flexible in nature. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 460–471, 2013.     

Support vector machines for the classification and prediction of beta-turn types.

The support vector machines (SVMs) method is proposed because it can reflect the sequence-coupling effect for a tetrapeptide in not only a beta-turn or non-beta-turn, but also in different types of beta-turn. The results of the model for 6022 tetrapeptides indicate that the rates of self-consistency for beta-turn types I, I', II, II', VI and VIII and non-beta-turns are 99.92%, 96.8%, 98.02%, 97.75%, 100%, 97.19% and 100%, respectively. Using these training data, the rate of correct prediction by the SVMs for a given protein: rubredoxin (54 residues. 51 tetrapeptides) which includes 12 beta-turn type I tetrapeptides, 1 beta-turn type II tetrapeptide and 38 non-beta-turns reached 82.4%. The high quality of prediction of the SVMs implies that the formation of different beta-turn types or non-beta-turns is considerably correlated with the sequence of a tetrapeptide. The SVMs can save CPU time and avoid the overfitting problem compared with the neural network method.     

Conformational changes of the p53-binding cleft of MDM2 revealed by molecular dynamics simulations

Two 35-ns molecular dynamics simulations of both ligated [mouse double minute protein 2 (MDM2(p53))] and unligated (MDM2(apo)) structures of human MDM2 bound to the N-terminal domain of the tumor suppressor p53 have been performed. Analysis of the dynamics revealed that the most flexible region of MDM2 was the p53-binding cleft. When MDM2 was bound to p53, a wider and more stable topology of the cleft was obtained, while unligated MDM2 showed a narrower and highly flexible cleft. It was also found that the dynamics involved in the opening/closing motions were due to the movement of different domains of the protein, which is in agreement with recent experimental data. Considering our results, a mechanism in which p53 might be recognized and attached to MDM2 is proposed, and some implications on future directions for in silico anticancer drug design efforts are discussed. In summary, the observations made here would be very useful not only for better understanding of the biological implications of the MDM2 dynamics, but also for future efforts in anticancer drug design and discovery.     

Effect of molecular confinement on internal enzyme dynamics: frequency domain fluorometry and molecular dynamics simulation studies

The tryptophanyl emission decay of the mesophilic beta-galactosidase from Aspergillus oryzae free in buffer and entrapped in agarose gel is investigated as a function of temperature and compared to that of the hyperthermophilic enzyme from Sulfolobus solfataricus. Both enzymes are tetrameric proteins with a large number of tryptophanyl residues, so the fluorescence emission can provide information on the conformational dynamics of the overall protein structure rather than that of the local environment. The tryptophanyl emission decays are best fitted by bimodal Lorentzian distributions. The long-lived component is ascribed to close, deeply buried tryptophanyl residues with reduced mobility; the short-lived one arises from tryptophanyl residues located in more flexible external regions of each subunit, some of which are involved in forming the catalytic site. The center of both lifetime distribution components at each temperature increases when going from the free in solution mesophilic enzyme to the gel-entrapped and hyperthermophilic enzyme, thus indicating that confinement of the mesophilic enzyme in the agarose gel limits the freedom of the polypeptide chain. A more complex dependence is observed for the distribution widths. Computer modeling techniques are used to recognize that the catalytic sites are similar for the mesophilic and hyperthermophilic beta-galactosidases. The effect due to gel entrapment is considered in dynamic simulations by imposing harmonic restraints to solvent-exposed atoms of the protein with the exclusion of those around the active site. The temperature dependence of the tryptophanyl fluorescence emission decay and the dynamic simulation confirm that more rigid structures, as in the case of the immobilized and/or hyperthermophilic enzyme, require higher temperatures to achieve the requisite conformational dynamics for an effective catalytic action and strongly suggest a link between conformational rigidity and enhanced thermal stability.     

Conformational studies of human islet amyloid peptide using molecular dynamics and simulated annealing methods

Molecular dynamics simulations and simulated annealing in vacuum, model aqueous solution, and simulated membrane were used to analyze the conformational preferences of a segment spanning 20–29 residues of human islet amyloid polypeptide, [referred to as IAPP^H(20–29)]. Molecular dynamics simulations were conducted at 300 K on IAPP^H(20–29). The minimum energy conformers obtained in model aqueous solution and vacuum exhibited similar structures. Even in the absence of any constraints on peptide bonds, trans conformation was preferred consistently by all the peptide bonds. Analysis of the minimum energy conformers indicated that IAPP^H(20–29) showed a strong preference for turn structures in all the environments. These turn structures were stabilized by the formation of hydrogen bonds between the backbone amide and carbonyl groups. A good agreement was found between the results obtained from the molecular dynamics simulation and solid-state nmr experimental studies. © 1998 John Wiley & Sons, Inc. Biopoly 45: 9–20, 1998     

NMR study and molecular dynamics simulations of optimized beta-hairpin fragments of protein G

The stability and structure of several beta-hairpin peptide variants derived from the C-terminus of the B1 domain of protein G were investigated by a number of experimental and computational techniques. Our analysis shows that the structure and stability of this hairpin can be greatly affected by one or a few simple mutations. For example, removing an unfavorable charge near the N-terminus of the peptide (Glu42 to Gln or Thr) or optimization of the N-terminal charge-charge interactions (Gly41 to Lys) both stabilize the peptide, even in water. Furthermore, a simple replacement of a charged residue in the turn (Asp47 to Ala) changes the beta-turn conformation. Finally, we show that the effects of combining these single mutations are additive, suggesting that independent stabilizing interactions can be isolated and evaluated in a simple model system. Our results indicate that the structure and stability of this beta-hairpin peptide can be modulated in numerous ways and thus contributes toward a more complete understanding of this important model beta-hairpin as well as to the folding and stability of larger peptides and proteins.     

Thermodynamic and kinetic characterization of a beta-hairpin peptide in solution: an extended phase space sampling by molecular dynamics simulations in explicit water

The folding of the amyloidogenic H1 peptide MKHMAGAAAAGAVV taken from the syrian hamster prion protein is explored in explicit aqueous solution at 300 K using long time scale all-atom molecular dynamics simulations for a total simulation time of 1.1 mus. The system, initially modeled as an alpha-helix, preferentially adopts a beta-hairpin structure and several unfolding/refolding events are observed, yielding a very short average beta-hairpin folding time of approximately 200 ns. The long time scale accessed by our simulations and the reversibility of the folding allow to properly explore the configurational space of the peptide in solution. The free energy profile, as a function of the principal components (essential eigenvectors) of motion, describing the main conformational transitions, shows the characteristic features of a funneled landscape, with a downhill surface toward the beta-hairpin folded basin. However, the analysis of the peptide thermodynamic stability, reveals that the beta-hairpin in solution is rather unstable. These results are in good agreement with several experimental evidences, according to which the isolated H1 peptide adopts very rapidly in water beta-sheet structure, leading to amyloid fibril precipitates [Nguyen et al., Biochemistry 1995;34:4186-4192; Inouye et al., J Struct Biol 1998;122:247-255]. Moreover, in this article we also characterize the diffusion behavior in conformational space, investigating its relations with folding/unfolding conditions.     

Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis

A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human alpha-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-alpha-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the beta-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the beta-domain of alpha-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.     

Conformational and structural analysis of the equilibrium between single- and double-strand beta-helix of a D,L-alternating oligonorleucine

Alternating sequences of D and L residues in peptides are directly related to the formation of several kinds of regular helical conformations usually called beta-helices. The major feature of these structures is that they can be associated with the transmembrane ion-conducting channel activity in some natural antibacterial peptides. The study of alternating D,L synthetic peptides is critical to understand how factors such as surrounding media, main chain length, type of side chain and terminal groups, among others, can determine the adoption of a specific kind of beta-helix. Early studies pointed out that the peptides Boc-(D-NLeu-L-NLeu)(6)-D-MeNLe-L-Nl-D-Nl-L-Nl-OMe (Boc: tert-butyloxycarbonyl) and Boc-L-Nle-(D-Nle-L-Nle)(5)-D-MeNle-L-Nle-D-Nle-L-Nle-OMe adopt in chloroform a unique detectable conformation single beta(4.4)- and double beta(5.6) upward arrow downward arrow -helix, respectively. The influence of terminal groups on the final stable conformation of N-formylated peptides has been studied in this work. The initial basic NMR data analysis of a synthetic alternating D,L-oligopeptide with ten norleucines, N-methylated on the residue 7 and having HCO- and -OMe as terminal groups clearly indicates the coexistence of two different conformations in equilibrium. NMR data and molecular dynamics calculations point to a dimeric antiparallel beta-helix structure beta(5.6) upward arrow downward arrow for the main conformation. On the other hand, NMR data suggest a single beta-helix structure beta(4.4) for the second conformation. Finally, a thermodynamic analysis of the equilibrium between both conformations has been carried out by one-dimensional NMR measurements at ten different temperatures. The temperature at which 50% of dimer conformation is dissociated is 319 K. In addition, the dimer-monomer equilibrium curve obtained shows a DeltaG>0 for the whole range of studied temperatures, and its behavior can be considered similar to the thermodynamic denaturation protein processes.     

Understanding the roles of amino acid residues in tertiary structure formation of chignolin by using molecular dynamics simulation

Chignolin is a 10-residue peptide (GYDPETGTWG) that forms a stable beta-hairpin structure in water. However, its design template, GPM12 (GYDDATKTFG), does not have a specific structure. To clarify which amino acids give it the ability to form the beta-hairpin structure, we calculated the folding free-energy landscapes of chignolin, GPM12, and their chimeric peptides using multicanonical molecular dynamics (MD) simulation. Cluster analysis of the conformational ensembles revealed that the native structure of chignolin was the lowest in terms of free energy while shallow local minima were widely distributed in the free energy landscape of GPM12, in agreement with experimental observations. Among the chimeric peptides, GPM12(D4P/K7G) stably formed the same beta-hairpin structure as that of chignolin in the MD simulation. This was confirmed by nuclear magnetic resonance (NMR) spectroscopy. A comparison of the free-energy landscapes showed that the conformational distribution of the Asp3-Pro4 sequence was inherently biased in a way that is advantageous both to forming hydrogen bonds with another beta-strand and to initiating loop structure. In addition, Gly7 helps stabilize the loop structure by having a left-handed alpha-helical conformation. Such a conformation is necessary to complete the loop structure, although it is not preferred by other amino acids. Our results suggest that the consistency between the short-range interactions that determine the local geometries and the long-range interactions that determine the global structure is important for stable tertiary structure formation.     

Conformational space search of Neuromedin C using replica exchange molecular dynamics and molecular dynamics

The present study involves the utilization of replica exchange molecular dynamics (REMD) methodology to explore the conformational space of Neuromedin C (NMC) using implicit (REMD^implicit) and explicit (REMD^explicit) water models. Comparison of the structures obtained from these simulations indicate that REMD^explicit trajectory display a greater tendency to induce β‐turns and bent structures as compared to those obtained from the REMD^implicit simulation. Moreover, two additional MD trajectories performed using Langevin (MD^Lang) and Berendsen (MD^Berend) algorithms under generalized born (GB) solvent conditions were also suitably competent to sample similar kinds of conformations, although the extent of beta turns was low compared to those observed in REMD^explicit simulation. Finally, the comparison of results obtained from all the trajectories and those derived from the NMR studies of Ni(II) complex of NMC indicates that the REMD under explicit conditions is more efficient in sampling the conformations, and show good agreement with the experimental results. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational dynamics of a short antigenic peptide in its free and antibody bound forms gives insight into the role of β‐turns in peptide immunogenicity

Earlier immunological experiments with a synthetic 36‐residue peptide (75‐110) from Influenza hemagglutinin have been shown to elicit anti‐peptide antibodies (Ab) which could cross‐react with the parent protein. In this article, we have studied the conformational features of a short antigenic (Ag) peptide (⁹⁸YPYDVPDYASLRS¹¹⁰) from Influenza hemagglutinin in its free and antibody (Ab) bound forms with molecular dynamics simulations using GROMACS package and OPLS‐AA/L all‐atom force field at two different temperatures (293 K and 310 K). Multiple simulations for the free Ag peptide show sampling of ordered conformations and suggest different conformational preferences of the peptide at the two temperatures. The free Ag samples a conformation crucial for Ab binding (β‐turn formed by “DYAS” sequence) with greater preference at 310 K while, it samples a native‐like conformation with relatively greater propensity at 293 K. The sequence “DYAS” samples β‐turn conformation with greater propensity at 310 K as part of the hemagglutinin protein also. The bound Ag too samples the β‐turn involving “DYAS” sequence and in addition it also samples a β‐turn formed by the sequence “YPYD” at its N‐terminus, which seems to be induced upon binding to the Ab. Further, the bound Ag displays conformational flexibility at both 293 K and 310 K, particularly at terminal residues. The implications of these results for peptide immunogenicity and Ag–Ab recognition are discussed. Proteins 2015; 83:1352–1367. © 2015 Wiley Periodicals, Inc.     

Structure and hydration of the amylopectin trisaccharide building blocks--Synthesis, NMR, and molecular dynamics

To gain insight into the molecular details and hydration of amylopectin, the five constituting trisaccharides have been chemically synthesized as their methyl alpha-glycosides. All five trisaccharides were subjected to 950 MHz NMR spectroscopy for complete assignment and nanosecond molecular dynamics trajectories were calculated to study the structure and dynamics of the trisaccharides in aqueous solution. Systematic analysis of the simulation data revealed several examples of bridging water molecules playing an important role in the stabilization of specific amylopectin conformations, which was also supported by the experimental NMR data such as interresidue NOE's and heteronuclear scalar couplings between nuclei from neighboring residues. Although alpha-maltotriose, alpha-iso-maltotriose, alpha-panose and alpha-isopanose are relatively well characterized structures, the study also includes one less characterized trisaccharide with the structure alphaGlcp(1-->4)alphaGlcp(1-->6)alphaGlcp. This trisaccharide, tentatively labelled alpha-forkose, is located at the branch point of amylopectin, forking the amylopectin into two strands that align into double-helical segments. The results show that the conformation of alpha-forkose takes a natural bend form which fits well into the structure of the double-helical segment of amylopectin. As the only trisaccharide in this study the structure of alpha-forkose is not significantly influenced by the hydration. In contrast, alpha-isopanose takes a restricted, but rather extended form due to an exceptionally strong localized water density. The two homo-linkage oligomers, alpha-maltotriose and alpha-iso-maltotriose, showed to be the most extended and the most flexible trimers, respectively, providing regular structure for crystalline domains and maximum linker flexibility for amorphous domains.     

Conformational investigation of alpha,beta-dehydropeptides. XVI. Beta-turn tendency in Ac-Pro-DeltaXaa-NHMe: crystallographic and theoretical studies.

The crystal structures of two diastereomeric alpha,beta-dehydrobutyrine peptides Ac-Pro-(Z)-DeltaAbu-NHMe (I) and Ac-Pro-(E)-DeltaAbu-NHMe (II) have been determined. Both dehydropeptides adopt betaI-turn conformation characterized by the pairs of (phi(i+1), psi(i+1)) and (phi(i+2), psi(i+2)) angles as -66, -19, -97, 11 degrees for I and -59, -27, -119, 29 degrees for II. In each peptide, the betaI turn is stabilized by (i + 3) --> i intramolecular hydrogen bonds with N...O distance of 3.12 A for I and 2.93 A for II. These structures have been compared to the crystal structures of homologous peptides Ac-Pro-DeltaVal-NHMe and Ac-Pro-DeltaAla-NHMe. Theoretical analyses by DFT/B3LYP/6-31 + G** method of conformers formed by these four peptides and by the saturated peptide Ac-Pro-Ala-NHMe revealed that peptides with a (Z) substituent at the C(beta) (i+2) atom of dehydroamino acid, i.e. Ac-Pro-DeltaVal-NHMe and Ac-Pro-(Z)-DeltaAbu-NHMe, predominantly form beta turns, both in vacuo and in polar environment. The tendency to adopt beta-turn conformation is much weaker for the peptides lacking the (Z) substituent, Ac-Pro-(E)-DeltaAbu-NHMe and Ac-Pro-DeltaAla-NHMe. The latter adopts a semi-extended or an extended conformation in every polar environment, including a weakly polar solvent. The saturated peptide Ac-Pro-Ala-NHMe in vacuo prefers a beta-turn conformation, but in polar environment the differences between various conformers are small. The role of pi-electron correlation and intramolecular hydrogen bonds interaction in stabilizing the hairpin structures are discussed.     

Structural characterization of human elastin derived peptides containing the GXXP sequence

The degradation of elastin, the insoluble biopolymer of tropoelastin, can lead to the production of small peptides. These elastin-derived peptides (EDPs) are playing a key role in cellular behavior within the extracellular matrix, showing a great variety of biological effects such as chemotaxis, stimulation of cell proliferation, ion flux modifications, vasorelaxation, and inflammatory enzymes secretion. It has also been demonstrated recently that EDPs containing the GXXPG motif could induce pro-MMP1 and pro-MMP3 upregulation. Elastolysis could then cause collagen degradation and play an important role in the aging process. Many experimental studies have been devoted to EDPs, but their structure/activity relationships are not well elucidated yet. However, the assumption that their active conformation is a type VIII beta-turn on GXXP was highly suggested on the basis of predictive statistical calculations. Investigation of the EDPs three-dimensional (3D) structure would provide useful information for drug-design strategies to propose specific inhibitors. The work presented here reports theoretical results obtained from molecular dynamics simulations performed over 128 human EDPs containing the GXXP motif. We show that all the peptides, for which the central residues are not glycines, adopt a canonical (or very close to) type VIII beta-turn structure on the GXXP sequence. Amino acids surrounding this motif are also important for the structural behavior. Any residue located before the GXXP motif (XGXXP) increases the beta-turn stabilization, whereas the residue located after GXXP (GXXPX) has no significant structural effect. Moreover, we show their biological activity can be correlated with their ability to exhibit a type VIII beta-turn conformation.     

Folding and stability of the three-stranded beta-sheet peptide Betanova: insights from molecular dynamics simulations

The dynamics of the three-stranded beta-sheet peptide Betanova has been studied at four different temperatures (280, 300, 350, and 450 K by molecular dynamics simulation techniques, in explicit water. Two 20-ns simulations at 280 K indicate that the peptide remains very flexible under "folding" conditions sampling a range of conformations that together satisfy the nuclear magnetic resonance (NMR)-derived experimental constraints. Two simulations at 300 K (above the experimental folding temperature) of 20 ns each show partial formation of "native"-like structure, which also satisfies most of the NOE constraints at 280 K. At higher temperature, the presence of compact states, in which a series of hydrophobic contacts remain present, are observed. This is consistent with experimental observations regarding the role of hydrophobic contacts in determining the peptide's stability and in initiating the formation of turns and loops. A set of different structures is shown to satisfy NMR-derived distance restraints and a possible mechanism for the folding of the peptide into the NMR-determined structure is proposed.     

Simulation of pH-dependent edge strand rearrangement in human beta-2 microglobulin

Amyloid fibrils formed from unrelated proteins often share morphological similarities, suggesting common biophysical mechanisms for amyloidogenesis. Biochemical studies of human beta-2 microglobulin (beta2M) have shown that its transition from a water-soluble protein to insoluble aggregates can be triggered by low pH. Additionally, biophysical measurements of beta2M using NMR have identified residues of the protein that participate in the formation of amyloid fibrils. The crystal structure of monomeric human beta2M determined at pH 5.7 shows that one of its edge beta-strands (strand D) adopts a conformation that differs from other structures of the same protein obtained at higher pH. This alternate beta-strand arrangement lacks a beta-bulge, which may facilitate protein aggregation through intermolecular beta-sheet association. To explore whether the pH change may yield the observed conformational difference, molecular dynamics simulations of beta2M were performed. The effects of pH were modeled by specifying the protonation states of Asp, Glu, and His, as well as the C terminus of the main chain. The bulged conformation of strand D is preferred at medium pH (pH 5-7), whereas at low pH (pH < 4) the straight conformation is observed. Therefore, low pH may stabilize the straight conformation of edge strand D and thus increase the amyloidogenicity of beta2M.     

Molecular dynamics simulations of alanine rich beta-sheet oligomers: Insight into amyloid formation

The aggregation observed in protein conformational diseases is the outcome of significant new beta-sheet structure not present in the native state. Peptide model systems have been useful in studies of fibril aggregate formation. Experimentally, it was found that a short peptide AGAAAAGA is one of the most highly amyloidogenic peptides. This peptide corresponds to the Syrian hamster prion protein (ShPrP) residues 113-120. The peptide was observed to be conserved in all species for which the PrP sequence has been determined. We have simulated the stabilities of oligomeric AGAAAAGA and AAAAAAAA (A8) by molecular dynamic simulations. Oligomers of both AGAAAAGA and AAAAAAAA were found to be stable when the size is 6 to 8 (hexamer to octamer). Subsequent simulation of an additional alpha-helical AAAAAAAA placed on the A8-octamer surface has revealed molecular events related to conformational change and oligomer growth. Our study addresses both the minimal oligomeric size of an aggregate seed and the mechanism of seed growth. Our simulations of the prion-derived 8-residue amyloidogenic peptide and its variant have indicated that an octamer is stable enough to be a seed and that the driving force for stabilization is the hydrophobic effect.     

Understanding the acylation mechanisms of active-site serine penicillin-recognizing proteins: a molecular dynamics simulation study

Beta-lactam antibiotics inhibit enzymes involved in the last step of peptidoglycan synthesis. These enzymes, also identified as penicillin-binding proteins (PBPs), form a long-lived acyl-enzyme complex with beta-lactams. Antibiotic resistance is mainly due to the production of beta-lactamases, which are enzymes that hydrolyze the antibiotics and so prevent them reaching and inactivating their targets, and to mutations of the PBPs that decrease their affinity for the antibiotics. In this study, we present a theoretical study of several penicillin-recognizing proteins complexed with various beta-lactam antibiotics. Hybrid quantum mechanical/molecular mechanical potentials in conjunction with molecular dynamics simulations have been performed to understand the role of several residues, and pK(a) calculations have also been done to determine their protonation state. We analyze the differences between the beta-lactamase TEM-1, the membrane-bound PBP2x of Streptococcus pneumoniae, and the soluble DD-transpeptidase of Streptomyces K15.