Molecular dynamics simulations of peptides containing an unnatural amino acid: dimerization, folding, and protein binding

We have performed molecular dynamics (MD) simulations to study the dimerization, folding, and binding to a protein of peptides containing an unnatural amino acid. NMR studies have shown that the substitution of one residue in a tripeptide beta-strand by the unnatural amino acid Hao (5-HO2CCONH-2-MeO-C6H3-CO-NHNH2) modifies the conformational flexibility of the beta-strand and the hydrogen-bonding properties of its two edges: The number of hydrogen-bond donors and acceptors increases at one edge, whereas at the other, they are sterically hindered. In simulations in chloroform, the Hao-containing peptide 9 (i-PrCO-Phe-Hao-Val-NHBu) forms a beta-sheet-like hydrogen-bonded dimer, in good agreement with the available experimental data. Addition of methanol to the solution induces instability of this beta-sheet, as confirmed by the experiments. MD simulations also reproduce the folding of the synthetic peptide 1a (i-PrCO-Hao-Ut-Phe-Ile-Leu-NHMe) into a beta-hairpin-like structure in chloroform. Finally, the Hao-containing peptide, Ac-Ala-Hao-Ala-NHMe, is shown to form a stable complex with the Ras analogue, Rap1 A, in water at room temperature. Together with the available experimental data, these simulation studies indicate that Hao-containing peptides may serve as inhibitors of beta-sheet interactions between proteins.     

Molecular simulation of the effects of alcohols on peptide structure

The effects of alcohols on local protein structure have been simulated using computational approaches and model peptides. Molecular simulations were carried out on a 7-residue peptide created in both an extended conformation and an alpha-helix to explore alcohol-induced changes in peptide structure. It was assumed that alcohols hydrogen bond at peptide carbonyl groups with an optimum geometry and compete with water molecules at these site. Energy minimization of the peptide/alcohol assemblies revealed that alcohols induced a twist in the peptide backbone as a function of (1) the methylene chain length, (2) the hydrogen-bond geometry, (3) halogenation of the molecule, (4) concentration, and (5) the dielectric constant. The rank ordering of the potencies of the alcohols was hexafluoroisopropanol > trifluoroethanol approximately pentanol > butanol > ethanol > methanol. Helix destabilization by cosolvent was measured by examining the hydrogen-bond lengths in peptide structures that resulted from a combination of energy minimization and molecular dynamics simulations. Destabilization was also found to be dependent upon the chemical nature of the alcohol and the hydrogen-bond geometry. The data suggest that alcohols at low concentrations affect protein structure mainly through a combination of hydrogen-bonding and hydrophobic interactions that are influenced by the properties of the solvent.     

Ester-linked hen egg white lysozyme shows a compact fold in a molecular dynamics simulation - possible causes and sensitivity of experimentally observable quantities to structural changes maintaining this compact fold

Prediction and understanding of the folding and stability of the 3D structure of proteins is still a challenge. The different atomic interactions, such as non polar contacts and hydrogen bonding, are known but their exact relative weights and roles when contributing to protein folding and stability are not identified. Initiated by a previous molecular dynamics simulation of fully ester-linked hen egg white lysozyme (HEWL), which showed a more compact fold of the ester-linked molecule compared to the native one, three variants of this protein are analyzed in the present study. These are 129-residue native HEWL, partly ester-linked HEWL, in which only 34 peptide linkages that are not involved in the helical or β-strand parts of native HEWL were replaced by ester linkages, and fully (126 residues) ester-linked HEWL. Native and partly ester-linked HEWL showed comparable behaviour, whereas fully ester-linked HEWL could not maintain the native secondary structure of HEWL in the simulation and adopted a more compact fold. The conformational changes were analyzed by comparing simulation averaged values of quantities that can be measured by NMR, such as (1)H-(15)N backbone order parameters, residual dipolar couplings, proton-proton NOE distances and (3)J-couplings with the corresponding values derived from experimental NMR data for native HEWL. The information content of the latter appeared to be insufficient to detect the local conformational rearrangements upon esterification of the loop regions of the protein. For fully ester-linked HEWL, a significantly reduced agreement was observed. Upon esterification, the backbone-side chain and side chain-side chain hydrogen-bonding pattern of HEWL changes to maintain its compactness and thus the structural stability of the ester-linked lysozymes.     

Free energy of helix propagation in short polyalanine chains determined from peptide growth simulations of La3+-binding model peptides. Comparison with experimental data

Molecular dynamics (MD) is, at present, a unique tool making it possible to study, at the atomic level, conformational transitions in peptides and proteins. Nevertheless, because MD calculations are always based on a more or less approximate physical model, using a set of approximate parameters, their reliability must be tested by comparison with experimental data. Unfortunately, it is very difficult to find a peptide system in which conformational transitions can be studied both experimentally and using MD simulations so that a direct comparison of the results obtained in both ways could be made. Such a system, containing a rigid alpha-helix nucleus stabilized by La(3+) coordination to a 12-residue sequence taken from an EF-hand protein has recently been used to determine experimentally the helix propagation parameters in very short polyalanine segments (Goch et al. (2003) Biochemistry 42: 6840-6847). The same parameters were calculated here for the same peptide system using the peptide growth simulation method with, alternatively, charmm 22 and cedar potential energy functions. The calculated free energies of the helix-coil transition are about two times too large for cedar and even three times too large for charmm 22, as compared with the experimental values. We suggest that these discrepancies have their origin in the incorrect representation of unfolded peptide backbone in solution by the molecular mechanics force fields.     

Molecular dynamics simulation of glycoprotein-glycans of immunoglobulin G and Immunoglobulin M

Glycoprotein-glycans have recently been implicated to play a variety of functional roles. The same glycan chain have been found complexed with proteins of diverse functions. In this article two such glycan chains found attached to Fc regions of immunoglobulin G and immunoglobulin M have been studied. An extensive simulated annealing procedure have been adopted to arrive at a low-energy minimum of the two oligosaccharides. Molecular dynamics simulations have been performed to study the flexibility of the glycosidic linkages. It was found that both glycan chains can undergo conformational transitions and adopt folded and extended conformations. The two β(1–2) linkages of complex-type glycan had been found to prefer different conformational regime and the terminal fucose linked to the GlcNAc residue drastically modifies the GlcNAc β(1–4)GlcNAc linkage conformation. In the high-mannose type glycan chain α(1–3) linkages can induce flexibility in addition to the α(1–6) linkages. The results have been compared with recent experimental nmr and fluorescence energy transfer data. © 1998 John Wiley & Sons, Inc. Biopoly 45: 177–190, 1998     

Methods of NMR structure refinement: molecular dynamics simulations improve the agreement with measured NMR data of a C-terminal peptide of GCN4-p1

The C-terminal trigger sequence is essential in the coiled-coil formation of GCN4-p1; its conformational properties are thus of importance for understanding this process at the atomic level. A solution NMR model structure of a peptide, GCN4p16–31, encompassing the GCN4-p1 trigger sequence was proposed a few years ago. Derived using a standard single-structure refinement protocol based on 172 nuclear Overhauser effect (NOE) distance restraints, 14 hydrogen-bond and 11 ϕ torsional-angle restraints, the resulting set of 20 NMR model structures exhibits regular α-helical structure. However, the set slightly violates some measured NOE bounds and does not reproduce all 15 measured ³J(H_N-H_Cα)-coupling constants, indicating that different conformers of GCN4p16–31 might be present in solution. With the aim to resolve structures compatible with all NOE upper distance bounds and ³J-coupling constants, we executed several structure refinement protocols employing unrestrained and restrained molecular dynamics (MD) simulations with two force fields. We find that only configurational ensembles obtained by applying simultaneously time-averaged NOE distance and ³J-coupling constant restraining with either force field reproduce all the experimental data. Additionally, analyses of the simulated ensembles show that the conformational variability of GCN4p16–31 in solution admitted by the available set of 187 measured NMR data is larger than represented by the set of the NMR model structures. The conformations of GCN4p16–31 in solution differ in the orientation not only of the side-chains but also of the backbone. The inconsistencies between the NMR model structures and the measured NMR data are due to the neglect of averaging effects and the inclusion of hydrogen-bond and torsional-angle restraints that have little basis in the primary, i.e. measured NMR data.     

The flexibility of the LeaLex Tumor Associated Antigen central fragment studied by systematic and stochastic searches as well as dynamic simulations

The solution conformational behavior of the Tumor-Associated Carbohydrate Antigen Le^aLe^x central fragment: methyl α-l-fucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-d-glucopyranosyl-(1→3)-β-d-galactopyranoside was studied using three computational methods: a rigid systematic search as implemented in Sybyl, a stochastic search as implemented in MOE2004, and dynamics simulations using the SANDER module of AMBER9. Our results illustrate the complementarity of these methods to identify energetically relevant conformations and flexible linkages. In particular, the β-GlcNAc-(1→3)-Gal linkage was shown to be extremely flexible adopting a wide range of orientations around two energy minima. The modeling results were validated by comparison of theoretical distances, derived from the simulations, with experimental measurements obtained from 1D selective ROESY buildup curves on the synthetic fragment.     

Molecular dynamics simulations of a cyclic-beta-(1-->2) glucan containing an alpha-(1-->6) linkage as a 'molecular alleviator' for the macrocyclic conformational strain

The conformational preferences of a cyclic osmoregulated periplasmic glucan of Ralstonia solanacearum (OPGR), which is composed of 13 glucose units and linked entirely via beta-(1-->2) linkages excluding one alpha-(1-->6) linkage, were characterized by molecular dynamics simulations. Of the three force fields modified for carbohydrates that were applied to select a suitable one for the cyclic glucan, the carbohydrate solution force field (CSFF) was found to most accurately simulate the cyclic molecule. To determine the conformational characteristics of OPGR, we investigated the glycosidic dihedral angle distribution, fluctuation, and the potential energy of the glucan and constructed hypothetical cyclic (CYS13) and linear (LINEAR) glucans. All beta-(1-->2)-glycosidic linkages of OPGR adopted stable conformations, and the dihedral angles fluctuated in this energy region with some flexibility. However, despite the inherent flexibility of the alpha-(1-->6) linkage, the dihedral angles have no transition and are more rigid than that in a linear glucan. CYS13, which consists of only beta-(1-->2) linkages, is somewhat less flexible than other glycans, and one of its linkages adopts a higher energy conformation. In addition, the root-mean-square fluctuation of this linkage is lower than that of other linkages. Furthermore, the potential energy of glucans increases in the order of LINEAR, OPGR, and CYS13. These results provide evidence of the existence of conformational constraints in the cyclic glucan. The alpha-(1-->6)-glycosidic linkage can relieve this constraint more efficiently than the beta-(1-->2) linkage. The conformation of OPGR can reconcile the tendency for individual glycosidic bonds to adopt energetically favorable conformations with the requirement for closure of the macrocyclic ring by losing the inherent flexibility of the alpha-(1-->6)-glycosidic linkage.     

The role of alpha-, 3(10)-, and pi-helix in helix-->coil transitions

The conformational equilibrium between 3(10)- and alpha-helical structure has been studied via high-resolution NMR spectroscopy by Millhauser and coworkers using the MW peptide Ac-AMAAKAWAAKA AAARA-NH2. Their 750-MHz nuclear Overhauser effect spectroscopy (NOESY) spectra were interpreted to reflect appreciable populations of 3(10)-helix throughout the peptide, with the greatest contribution at the N and C termini. The presence of simultaneous alphaN(i,i + 2) and alphaN(i,i + 4) NOE cross-peaks was proposed to represent conformational averaging between 3(10)- and alpha-helical structures. In this study, we describe 25-nsec molecular dynamics simulations of the MW peptide at 298 K, using both an 8 A and a 10 A force-shifted nonbonded cutoff. The ensemble averages of both simulations are in reasonable agreement with the experimental helical content from circular dichroism (CD), the (3)J(HNalpha) coupling constants, and the 57 observed NOEs. Analysis of the structures from both simulations revealed very little formation of contiguous i --> i + 3 hydrogen bonds (3(10)-helix); however, there was a large population of bifurcated i --> i + 3 and i --> i + 4 alpha-helical hydrogen bonds. In addition, both simulations contained considerable populations of pi-helix (i --> i + 5 hydrogen bonds). Individual turns formed over residues 1-9, which we predict contribute to the intensities of the experimentally observed alphaN(i,i + 2) NOEs. Here we show how sampling of both folded and unfolded structures can provide a structural framework for deconvolution of the conformational contributions to experimental ensemble averages.     

A molecular dynamics study of the effect of glycosidic linkage type in the hemicellulose backbone on the molecular chain flexibility

The macromolecular conformation of the constituent polysaccharides in lignocellulosic biomass influences their supramolecular interactions, and therefore their function in plants and their performance in technical products. The flexibility of glycosidic linkages from the backbone of hemicelluloses was studied by evaluating the conformational freedom of the φ and ψ dihedral angles using molecular dynamic simulations, additionally selected molecules were correlated with experimental data by nuclear magnetic resonance spectroscopy. Three types of β‐(1→4) glycosidic linkages involving the monosaccharides (Glcp, Xylp and Manp) present in the backbone of hemicelluloses were defined. Different di‐ and tetrasaccharides with combinations of such sugar monomers from hemicelluloses were simulated, and free energy maps of the φ – ψ space and hydrogen‐bonding patterns were obtained. The glycosidic linkage between Glc‐Glc or Glc‐Man (C‐type) was the stiffest with mainly one probable conformation; the linkage from Man‐Man or Man‐Glc (M‐type) was similar but with an increased probability for an alternative conformation making it more flexible, and the linkage between two Xyl‐units (X‐type) was the most flexible with two almost equally populated conformations. Glycosidic linkages of the same type showed essentially the same conformational space in both disaccharides and in the central region of tetrasaccharides. Different probabilities of glycosidic linkage conformations in the backbone of hemicelluloses can be directly estimated from the free energy maps, which to a large degree affect the overall macromolecular conformations of these polymers. The information gained contributes to an increased understanding of the function of hemicelluloses both in the cell wall and in technical products.     

Effects of base substitutions in an RNA hairpin from molecular dynamics and free energy simulations

Contributions of individual interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using base substitutions. The G in the first tetraloop position was replaced by inosine (I) or adenosine (A), and the G in the C-G basepair closing the tetraloop was replaced by I. These substitutions eliminate particular hydrogen bonds proposed in the nuclear magnetic resonance model of the GCAA tetraloop. Molecular dynamics simulations of the GCAA tetraloop in aqueous solvent displayed a well-defined hydrogen pattern between the first and last loop nucleotides (G and A) stabilized by a bridging water molecule. Substitution of G-->I in the basepair closing the tetraloop did not significantly influence the loop structure and dynamics. The ICAA loop maintained the overall structure, but displayed variation in the hydrogen-bond network within the tetraloop itself. Molecular dynamics simulations of the ACAA loop led to conformational heterogeneity of the resulting structures. Changes of hairpin formation free energy associated with substitutions of individual bases were calculated by the free energy perturbation method. The calculated decrease of the hairpin stability upon G-->I substitution in the C-G basepair closing the tetraloop was in good agreement with experimental thermodynamic data. Our theoretical estimates for G-->I and G-->A mutations located in the tetraloop suggest larger loop destabilization than corresponding experimental results. The extent of conformational sampling of the structures resulting from base substitutions and its impact on the calculated free energy was discussed.     

Combined use of molecular dynamics simulations and NMR to explore peptide bond isomerization and multiple intramolecular hydrogen-bonding possibilities in a cyclic pentapeptide, cyclo(Gly-Pro-D-Phe-Gly-Val).

The conformational behavior of a model cyclic pentapeptide--cyclo(Gly-L-Pro-D-Phe-Gly-L-Val)--has been explored through the combined use of in vacuo molecular dynamics simulations and a range of nmr experiments (preceding paper). The molecular dynamics analysis suggests that, despite the conformational constraints imposed by formation of the pentapeptide cycle, this pentapeptide undergoes conformational transitions between various hydrogen-bonded conformations, characterized by low energy barriers. An inverse gamma turn with Pro in position i + 1 and a gamma turn with D-Phe in position i + 1 are two alternatives occurring frequently. Like other DLDDL cyclic pentapeptides, cyclo(Gly-Pro-D-Phe-Gly-Val) is also stabilized by an inverse gamma-turn structure with the beta-branched Val residue in position i + 1, and this hydrogen bond is retained in the different conformational families. The gamma-turn around D-Phe3 and the inverse gamma turn around Val5 are consistent with the nmr observations. 3JNH-CH alpha coupling constants of the all-trans forms were calculated from one of the molecular dynamics trajectories and are comparable to nmr experimental data, suggesting that the conformational states visited during the simulation are representative of the conformational distribution in solution. In addition to the equilibrium among various hydrogen-bonded all-trans conformers, the observation in nmr spectra of two sets of resonances for all peptide protons indicated a slow conformational interconversion of the Gly-Pro peptide bond between trans and cis isomers. The activation energy between these two conformers was determined experimentally by magnetization transfer and was calculated by high temperature constrained molecular dynamics simulation. Both methods yield a free energy of activation of ca. 20 kcal/mol. Furthermore, the free energy of activation is dependent on the direction of rotation of the Gly-Pro peptide bond.     

Conformational studies of -glucans

A study of the effect of linkage on the possible conformations of di-and polysaccharides of α-D -glucose and also the probable intramolecular hydrogen bonds has been made. The differences in the nature of linkage is shown to effect the energetically preferred conformations; (1 → 2) linkages lead only to righthanded helical conformations, (1 → 3) linkages lead to extended as well as both left and righthanded helical conformations; (1 → 4) linkages lead to both right-and lefthanded wide helical conformations. The possible hydrogen bonds between adjacent residues are also dependent on the nature of the linkage. A comparison of the conformational data of α-D -glucans with those of β-D -glucans has indicated that the favored conformations and hydrogen bonds between contiguous residues in the chain are influenced by the configuration at the anomeric carbon atom in all the three types of polysaccharides. From the energy calculations a probable conformation (?M = ?10°, ψM = ?30°, ?N = ?23°, ψN = ?19°) has also been proposed for crystalline mycodextran in conformity with x-ray data. This conformation contains two types of hydrogen bonds between contiguous residues one between 0–2 and 0–3 atoms at (1 → 4) linkage and the other between 0?2 and 0–4 atoms at (1 → 3) linkage in the chain. The conformation of maltose unit (?10°,?30°) that is likely to occur in the crystalline mycodextran coincides with the minimum energy conformation of maltose.     

Investigations of the MceIJ-catalyzed posttranslational modification of the microcin E492 C-terminus: linkage of ribosomal and nonribosomal peptides to form "trojan horse" antibiotics

MceIJ is a two protein complex responsible for attachment of a C-glycosylated and linearized derivative of enterobactin, an iron scavenger (siderophore) and product of nonribosomal peptide synthetase machinery, to the C-terminal serine residue of microcin E492 (MccE492), an 84 aa ribosomal antibiotic peptide produced by Klebsiella pneumoniae RYC492. The MceIJ-catalyzed formation of the glycosyl ester linkage between MccE492 and the siderophore requires ATP and Mg(II) as cofactors. This work addresses the ATP utilization, mechanism of C-terminal carboxylate activation, and substrate scope of MceIJ. Formation of the ribosomal peptide-nonribosomal peptide linkage between the MccE492 C-terminal decapeptide and monoglycosylated enterobactin (MGE) requires cleavage of the alpha,beta bond of ATP and formation of a putative peptidyl-CO-AMP intermediate. Attack of the peptidyl-CO-AMP carbonyl by the deprotonated C4' hydroxyl of the glucose moiety forms a glycosyl ester linkage with release of AMP. Site-directed mutagenesis of the three cysteines and five histidines in MceI to alanines reveals that these residues are not required structurally or catalytically. MceIJ recognizes all glycosylated enterobactin derivatives formed by the MccE492 gene cluster members MceC ( C-glycosyltransferase) and MceD (esterase) in vitro and a MGE derivative lacking the C6' hydroxyl moiety. The protein complex also accepts and modifies the C-terminal decapeptide substrate fragments of the structurally related microcins H47, I47, and M. MccE492 C-terminal decapeptides bearing fluorescein and biotin moieties on the N-terminus are also substrates for MceIJ, which provides a route for the chemoenzymatic synthesis of enterobactin conjugates with peptide linkages.     

Conformational analysis of an alpha-galactosyl trisaccharide epitope involved in hyperacute rejection upon xenotransplantation.

alpha-Galactosyl epitopes are carbohydrate structures bearing an alpha-Gal-(1-->3)-Gal terminus (alpha-Gal epitopes). The interaction of these epitopes on the surface of animal cells with anti alpha-Gal antibodies in human serum is believed to be the main cause in antibody-mediated hyperacute rejection in xenotransplantation. In this paper, conformational analysis of an N-linked alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp trisaccharide epitope was conducted in terms of each monosaccharide residue conformation, primary hydroxymethyl group configuration, and interglycosidic conformations. Selective 2D J-delta INEPT experiments have been carried out at three different temperatures to evaluate three-bond, long-range 13C-1H coupling constants for the crucial alpha-(1-->3) linkage. The NMR experimental data were complemented by theoretical calculations. The flexibility and dynamics of the trisaccharide have been studied by Metropolis Monte Carlo simulations. Ensemble-averaged three-bond, long-range 13C-1H coupling constants and nuclear Overhauser effects were in good agreement with the experimental data. The alpha-(1-->3) glycosidic linkage has shown a restricted flexibility as indicated by NMR spectroscopy and molecular modeling.     

Conformational variability of the synthetic peptide 129-141 of the mouse prion protein

The effect of solution conditions on the conformation of the peptide corresponding to residues 129-141 of the mouse prion protein has been examined by experimental and theoretical tools including circular dichroism, secondary structure predictions, and Molecular Dynamics simulations. The conformational properties of the peptide observed by CD confirm the prediction results: the peptide is chiefly random coil in water. The conformational sampling performed by Molecular Dynamics simulations in water also corroborates the flexibility of the peptide, in particular for the N-terminal part. We show, however, that the peptide samples hairpin conformations in one of several approximately 1-ns Molecular Dynamics simulations in water. Interestingly, the analysis of the CD spectra obtained in this study suggests the presence of beta-structure which, given the length of the peptide, can only consist in beta-hairpin. The peptide can also be induced to form a modest percentage of helical structure in the presence of organic cosolvents such as trifluoroethanol, or detergents such as sodium dodecyl sulfate and lysophosphatidylcholine. This result is different from that obtained for a homologous hamster fragment, which differs from the mouse sequence by the single substitution of Ile 139 to Met. Interestingly, this substitution is crucial for the barrier in the transmission of the prion disease between hamsters and mice.     

Stability and fluctuations of amide hydrogen bonds in a bacterial cytochrome c: a molecular dynamics study

Molecular dynamics (MD) simulations on a bacterial cytochrome c were performed to investigate the lifetime and fluctuations of backbone hydrogen bonds and to correlate these data with protection factors for hydrogen exchange measured by NMR spectroscopy (Bartalesi et al. in Biochemistry, 42:10923–10930, 2003). The MD simulations provide a consistent pattern in that long lifetimes of hydrogen bonds go along with small amplitude fluctuations. In agreement with experiments, differences in stability were found with a rather flexible N-terminal segment as compared with a more rigid C-terminal part. Protection factors of backbone hydrogen exchange correlate strongly with the number of contacts but also with hydrogen-bond occupancy, hydrogen-bond survival times, as well as the inverse of fluctuations of backbone atoms and hydrogen-bond lengths derived from MD simulation data. We observed a conformational transition in the C-terminal loop, and significant motion in the N-terminal loop, which can be interpreted as being the structural units involved in the onset of the protein unfolding process in agreement with experimental evidence on mitochondrial cytochrome c. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Gernot Kieseritzky and Giulia Morra both contributed equally to this work.     

Conformational flexibility of the MHC class I alpha1-alpha2 domain in peptide bound and free states: a molecular dynamics simulation study

Major histocompatibility complex class I proteins play a key role in the recognition and presentation of peptide antigens to the host immune system. The structure of various major histocompatibility complex class I proteins has been determined experimentally in complex with several antigenic peptides. However, the structure in the unbound (empty) form is not known. To study the conformational dynamics of the empty major histocompatibility complex class I molecule comparative molecular dynamics simulations have been performed starting from the crystal structure of a peptide bound class I peptide-binding domain in the presence and absence of a peptide ligand. Simulations including the bound peptide stayed close to the experimental start structure at both simulation temperatures (300 and 355 K) during the entire simulation of 26 ns. Several independent simulations in the absence of peptide indicate that the empty domain may not adopt a single defined conformation but is conformationally significantly more heterogeneous in particular within the alpha-helices that flank the peptide binding cleft. The calculated conformational dynamics along the protein chain correlate well with available spectroscopic data and with the observed site-specific sensitivity of the empty class I protein to proteolytic digestion. During the simulations at 300 K the binding region for the peptide N-terminus stayed close to the conformation in the bound state, whereas the anchor region for the C-terminus showed significantly larger conformational fluctuations. This included a segment at the beginning of the second alpha-helix in the domain that is likely to be involved in the interaction with the chaperone protein tapasin during the peptide-loading process. The simulation studies further indicate that peptide binding at the C- and N-terminus may follow different mechanisms that involve different degrees of induced conformational changes in the peptide-binding domain. In particular binding of the peptide C-terminus may require conformational stabilization by chaperone proteins during peptide loading.     

Backbone conformational flexibility of the lipid modified membrane anchor of the human N-Ras protein investigated by solid-state NMR and molecular dynamics simulation

The lipid modified human N-Ras protein, implicated in human cancer development, is of particular interest due to its membrane anchor that determines the activity and subcellular location of the protein. Previous solid-state NMR investigations indicated that this membrane anchor is highly dynamic, which may be indicative of backbone conformational flexibility. This article aims to address if a dynamic exchange between three structural models exist that had been determined previously. We applied a combination of solid-state nuclear magnetic resonance (NMR) methods and replica exchange molecular dynamics (MD) simulations using a Ras peptide that represents the terminal seven amino acids of the human N-Ras protein. Analysis of correlations between the conformations of individual amino acids revealed that Cys 181 and Met 182 undergo collective conformational exchange. Two major structures constituting about 60% of all conformations could be identified. The two conformations found in the simulation are in rapid exchange, which gives rise to low backbone order parameters and nuclear spin relaxation as measured by experimental NMR methods. These parameters were also determined from two 300 ns conventional MD simulations, providing very good agreement with the experimental data.