Helical content of a β(3)-octapeptide in methanol: molecular dynamics simulations explain a seeming discrepancy between conclusions derived from CD and NMR data

Connecting experimental observables with the underlying conformational ensemble is a long-standing problem in the structure determination of biomolecules. The simulations described in this article attempt to resolve a seeming discrepancy between the conformational features derived from measured NOE intensities, (3)J-coupling constants, and circular dichroism (CD) spectra for two β-peptides differing in a linker between two side-chains. Although both peptides are very similar in terms of the r(-6) averaged distances between atom pairs involved in the observed NOEs, the molecular dynamics trajectories suggest why the CD spectra show a greater 3(14)-helical propensity for the linked, cyclic peptide than for the linear one, whereas slightly more NMR NOE peaks are observed and assigned for the latter. The nine 100 ns unrestrained simulations show better agreement with the observed experimental data than the single conformations derived from the published NMR structures by additional energy minimization with the GROMOS force field. They show why the seemingly contradictory quantities obtained by NMR and CD spectroscopy can arise from a single conformational ensemble.     

RNA unrestrained molecular dynamics ensemble improves agreement with experimental NMR data compared to single static structure: a test case

Nuclear magnetic resonance (NMR) provides structural and dynamic information reflecting an average, often non-linear, of multiple solution-state conformations. Therefore, a single optimized structure derived from NMR refinement may be misleading if the NMR data actually result from averaging of distinct conformers. It is hypothesized that a conformational ensemble generated by a valid molecular dynamics (MD) simulation should be able to improve agreement with the NMR data set compared with the single optimized starting structure. Using a model system consisting of two sequence-related self-complementary ribonucleotide octamers for which NMR data was available, 0.3 ns particle mesh Ewald MD simulations were performed in the AMBER force field in the presence of explicit water and counterions. Agreement of the averaged properties of the molecular dynamics ensembles with NMR data such as homonuclear proton nuclear Overhauser effect (NOE)-based distance constraints, homonuclear proton and heteronuclear ¹H–³¹P coupling constant (J) data, and qualitative NMR information on hydrogen bond occupancy, was systematically assessed. Despite the short length of the simulation, the ensemble generated from it agreed with the NMR experimental constraints more completely than the single optimized NMR structure. This suggests that short unrestrained MD simulations may be of utility in interpreting NMR results. As expected, a 0.5 ns simulation utilizing a distance dependent dielectric did not improve agreement with the NMR data, consistent with its inferior exploration of conformational space as assessed by 2-D RMSD plots. Thus, ability to rapidly improve agreement with NMR constraints may be a sensitive diagnostic of the MD methods themselves.     

The beta-peptide hairpin in solution: conformational study of a beta-hexapeptide in methanol by NMR spectroscopy and MD simulation.

The structural and thermodynamic properties of a 6-residue beta-peptide that was designed to form a hairpin conformation have been studied by NMR spectroscopy and MD simulation in methanol solution. The predicted hairpin would be characterized by a 10-membered hydrogen-bonded turn involving residues 3 and 4, and two extended antiparallel strands. The interproton distances and backbone torsional dihedral angles derived from the NMR experiments at room temperature are in general terms compatible with the hairpin conformation. Two trajectories of system configurations from 100-ns molecular-dynamics simulations of the peptide in solution at 298 and 340 K have been analyzed. In both simulations reversible folding to the hairpin conformation is observed. Interestingly, there is a significant conformational overlap between the unfolded state of the peptide at each of the temperatures. As already observed in previous studies of peptide folding, the unfolded state is composed of a (relatively) small number of predominant conformers and in this case lacks any type of secondary-structure element. The trajectories provide an excellent ground for the interpretation of the NMR-derived data in terms of ensemble averages and distributions as opposed to single-conformation interpretations. From this perspective, a relative population of the hairpin conformation of 20% to 30% would suffice to explain the NMR-derived data. Surprisingly, however, the ensemble of structures from the simulation at 340 K reproduces more accurately the NMR-derived data than the ensemble from the simulation at 298 K, a question that needs further investigation.     

Solute-solvent interactions probed by intermolecular NOEs

Nuclear Overhauser effects arising from the interactions of spins of solvent molecules with spins of a solute should reveal the "exposure" of solute spins to collisions with solvent. Such intermolecular NOEs could, therefore, provide information regarding conformation or structure of the solute. Determinations of solute-solvent NOEs of 1,3-di-tert-butylbenzene in solvents composed of perfluoro-tert-butyl alcohol, tetramethylsilane, and carbon tetrachloride have been carried out. A crude, but apparently reliable, method for prediction of intermolecular solvent-solute NOEs based on hard (noninteracting) spheres was developed. Comparison of experimental to predicted NOEs indicates that tetramethylsilane interacts with the solute according to the model. By contrast, intermolecular NOE data indicate attractive interactions between the solute and perfluoro-tert-butyl alcohol. All NOE results and the corresponding predictions confirm that proton H2 of the solute is protected by the flanking tert-butyl groups from interactions with solvent molecules.     

Validation of the GROMOS 54A7 Force Field Regarding Mixed α/β‐Peptide Molecules

A molecular‐dynamics (MD) simulation study of two heptapeptides containing α‐ and β‐amino acid residues is presented. According to NMR experiments, the two peptides differ in dominant fold when solvated in MeOH: peptide 3 adopts predominantly β‐hairpin‐like conformations, while peptide 8 adopts a 14/15‐helical fold. The MD simulations largely reproduce the experimental data. Application of NOE atom? atom distance restraining improves the agreement with experimental data, but reduces the conformational sampling. Peptide 3 shows a variety of conformations, while still agreeing with the NOE and ³J‐coupling data, whereas the conformational ensemble of peptide 8 is dominated by one helical conformation. The results confirm the suitability of the GROMOS 54A7 force field for simulation or structure refinement of mixed α/β‐peptides in MeOH.     

Establishing effective simulation protocols for beta- and alpha/beta-peptides. II. Molecular mechanical (MM) model for a cyclic beta-residue

All-atom molecular mechanical (MM) force field parameters are developed for a cyclic beta-amino acid, amino-cyclo-pentane-carboxylic acid (ACPC), using a multi-objective evolutionary algorithm. The MM model is benchmarked using several short, ACPC-containing alpha/beta-peptides in water and methanol with SCC-DFTB (self consistent charge-density functional tight binding)/MM simulations as the reference. Satisfactory agreements are found between the MM and SCC-DFTB/MM results regarding the distribution of key dihedral angles for the tetra-alpha/beta-peptide in water. For the octa-alpha/beta-peptide in methanol, the MM and SCC-DFTB/MM simulations predict the 11- and 14/15-helical form as the more stable conformation, respectively; however, the two helical forms are very close in energy (2-4 kcal/mol) at both theoretical levels, which is also the conclusion from recent NMR experiments. As the first application, the MM model is applied to an alpha/beta-pentadeca-peptide in water with both explicit and implicit solvent models. The stability of the peptide is sensitive to the starting configuration in the explicit solvent simulations due to their limited length ( approximately 10-40 ns). Multiple ( approximately 20 x 20 ns) implicit solvent simulations consistently show that the 14/15-helix is the predominant conformation of this peptide, although substantially different conformations are also accessible. The calculated nuclear Overhauser effect (NOE) values averaged over different trajectories are consistent with experimental data, which emphasizes the importance of considering conformational heterogeneity in such comparisons for highly dynamical peptides.     

Spectroscopic evidence of beta-turn in N-glycated peptidomimetics related to leucine-enkephalin

The conformational differences caused by N-glycation of the amide bond in endogenous opioid pentapeptide leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu) have been explored in solution using FTIR spectroscopy, NMR and molecular modelling. The compounds studied include protected and unprotected enkephalin analogues N-alkylated at the second (Gly2) amino acid residue with a 6-deoxy-D-galactose moiety (1-3). Comparison of the amide I component bands in the FTIR spectra, measured in trifluoroethanol (TFE), CHCl3 and DMSO, revealed significant differences in the intensity as well as shifts in component band frequencies for glycopeptides 1-3. We found that only the FTIR spectrum of the fully protected compound 1 indicated the presence of a higher population of beta-turns, while the spectra of the partially protected and unprotected glycopeptides 2 and 3 reflected the dominance of unordered or open structures, with some low population of turns. The observed NOE connectivities in CDCl3 for both isomers of the fully protected compound 1, the all-trans one and another with Tyr1-Gly2 peptide bond in cis conformation, indicate the presence of a beta-like turn conformation. Molecular dynamics simulations of the glycopeptide 1 obtained by unconstrained energy minimization of trans- and cis-1 shows that one of trans form conformations is consistent with beta-turn whereas cis isomer has revealed less-compact turn.     

Conformational Dynamics of apo‐GlnBP Revealed by Experimental and Computational Analysis

The glutamine binding protein (GlnBP) binds l ‐glutamine and cooperates with its cognate transporters during glutamine uptake. Crystal structure analysis has revealed an open and a closed conformation for apo‐ and holo‐GlnBP, respectively. However, the detailed conformational dynamics have remained unclear. Herein, we combined NMR spectroscopy, MD simulations, and single‐molecule FRET techniques to decipher the conformational dynamics of apo‐GlnBP. The NMR residual dipolar couplings of apo‐GlnBP were in good agreement with a MD‐derived structure ensemble consisting of four metastable states. The open and closed conformations are the two major states. This four‐state model was further validated by smFRET experiments and suggests the conformational selection mechanism in ligand recognition of GlnBP.     

Application of paramagnetic NMR-validated molecular dynamics simulation to the analysis of a conformational ensemble of a branched oligosaccharide

Oligosaccharides of biological importance often exhibit branched covalent structures and dynamic conformational multiplicities. Here we report the application of a method that we developed, which combined molecular dynamics (MD) simulations and lanthanide-assisted paramagnetic NMR spectroscopy, to evaluate the dynamic conformational ensemble of a branched oligosaccharide. A lanthanide-chelating tag was attached to the reducing end of the branched tetrasaccharide of GM2 ganglioside to observe pseudocontact shifts as the source of long distance information for validating the conformational ensemble derived from MD simulations. By inspecting the results, the conformational space of the GM2 tetrasaccharide was compared with that of its nonbranched derivative, the GM3 trisaccharide.     

A Method to Explore Protein Side Chain Conformational Variability Using Experimental Data

Experimentally measured values of molecular properties or observables of biomolecules such as proteins are generally averages over time and space, which do not contain su?cient information to determine the underlying conformational distribution of the molecules in solution. The relationship between experimentally measured NMR ³J‐coupling values and the corresponding dihedral angle values is a particularly complicated case due to its nonlinear, multiple‐valued nature. Molecular dynamics (MD) simulations at constant temperature can generate Boltzmann ensembles of molecular structures that are free from a priori assumptions about the nature of the underlying conformational distribution. They suffer, however, from limited sampling with respect to time and conformational space. Moreover, the quality of the obtained structures is dependent on the choice of force ?eld and solvation model. A recently proposed method that uses time‐averaging with local‐elevation (LE) biasing of the conformational search provides an elegant means of overcoming these three problems. Using a set of side chain ³J‐coupling values for the FK506 binding protein (FKBP), we ?rst investigate the uncertainty in the angle values predicted theoretically. We then propose a simple MD‐based technique to detect inconsistencies within an experimental data set and identify degrees of freedom for which conformational averaging takes place or for which force ?eld parameters may be de?cient. Finally, we show that LE MD is the best method for producing ensembles of structures that, on average, ?t the experimental data.     

What induces pocket openings on protein surface patches involved in protein–protein interactions?

We previously showed for the proteins BCL-X_L, IL-2, and MDM2 that transient pockets at their protein–protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein–protein interfaces. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interaction of a tripeptide with cesium perfluorooctanoate micelles

The interaction of alanyl-phenylalanyl-alanine (Ala-Phe-Ala) with the micelles formed by cesium perfluorooctanoate (CsPFO) in water was studied in the isotropic phase by means of 1H NMR and by molecular dynamics (MD) simulations. Information on the location of the peptide was experimentally obtained from selective variations in Ala-Phe-Ala chemical shifts and from differential line broadening in the presence of the paramagnetic ion Mn2+. The peptide-micelle association constant was estimated analyzing the chemical shift variations of the most sensitive Ala-Phe-Ala resonances with the peptide concentration. MD simulations of Ala-Phe-Ala in the micellar environment confirmed the experimental observations, identifying the hydrogen bonding interactions of the different peptide moieties with the micelle, yielding a binding constant close to the experimental one. NOESY experiments suggest that the peptide in the micellar environment does not adopt a preferred conformation but is mainly unstructured. Details on the conformational behavior of the peptide in the micellar solution observed through MD were consistent with a different conformational equilibrium in the proximity of the micelle. Information on Ala-Phe-Ala dynamics was obtained from 1H T1 data and compared to MD simulation results on the overall tumbling motion.     

A simple method to predict protein flexibility using secondary chemical shifts

Protein motions play a critical role in many biological processes, such as enzyme catalysis, allosteric regulation, antigen-antibody interactions, and protein-DNA binding. NMR spectroscopy occupies a unique place among methods for investigating protein dynamics due to its ability to provide site-specific information about protein motions over a large range of time scales. However, most NMR methods require a detailed knowledge of the 3D structure and/or the collection of additional experimental data (NOEs, T1, T2, etc.) to accurately measure protein dynamics. Here we present a simple method based on chemical shift data that allows accurate, quantitative, site-specific mapping of protein backbone mobility without the need of a three-dimensional structure or the collection and analysis of NMR relaxation data. Further, we show that this chemical shift method is able to quantitatively predict per-residue RMSD values (from both MD simulations and NMR structural ensembles) as well as model-free backbone order parameters.     

Paramagnetic-based NMR restraints lift residual dipolar coupling degeneracy in multidomain detergent-solubilized membrane proteins

Residual dipolar couplings (RDCs) are widely used as orientation-dependent NMR restraints to improve the resolution of the NMR conformational ensemble of biomacromolecules and define the relative orientation of multidomain proteins and protein complexes. However, the interpretation of RDCs is complicated by the intrinsic degeneracy of analytical solutions and protein dynamics that lead to ill-defined orientations of the structural domains (ghost orientations). Here, we illustrate how restraints from paramagnetic relaxation enhancement (PRE) experiments lift the orientational ambiguity of multidomain membrane proteins solubilized in detergent micelles. We tested this approach on monomeric phospholamban (PLN), a 52-residue membrane protein, which is composed of two helical domains connected by a flexible loop. We show that the combination of classical solution NMR restraints (NOEs and dihedral angles) with RDC and PRE constraints resolves topological ambiguities, improving the convergence of the PLN structural ensemble and giving the depth of insertion of the protein within the micelle. The combination of RDCs with PREs will be necessary for improving the accuracy and precision of membrane protein conformational ensembles, where three-dimensional structures are dictated by interactions with the membrane-mimicking environment rather than compact tertiary folds common in globular proteins.     

Structures of protein-protein complexes are docked using only NMR restraints from residual dipolar coupling and chemical shift perturbations

NMR structures of protein-protein and protein-ligand complexes rely heavily on intermolecular NOEs. Recent work has shown that if no significant conformational changes occur upon complex formation residual dipolar coupling can replace most of the NOE restraints in protein-protein complexes, while restraints derived from chemical shift perturbations can largely replace intermolecular NOEs in protein-ligand structures. By combining restraints from chemical shift perturbations with orientation restraints derived from measurements of residual dipolar couplings, we show that the structure of the EIN-HPr complex can be calculated without NOE restraints. The final structure, built from the crystal structures of EIN and HPr in their uncomplexed form and docked only with NMR restraints, places HPr within 2.5 A of the position determined from the mean NMR structure of the complex.     

Biophysical Evaluation and In Vitro Controlled Release of Two Isomeric Adamantane Phenylalkylamines with Antiproliferative/Anticancer and Analgesic Activity

The aqueous dissolution profile of the isomeric synthetic adamantane phenylalkylamine hydrochlorides I and II was probed. These adducts have shown significant antiproliferative/anticancer activity associated with an analgesic profile against neuropathic pain. They are both devoid of toxic effects and show appreciable enzymatic human plasma stability. The structures of these two compounds have been elucidated using 2D NMR experiments, which were used to study their predominant conformations. Compound II’s scaffold appeared more flexible, as shown by the NOE spatial interactions between the alkyl bridge chain, the aromatic rings, and the adamantane nucleus. Conversely, compound I appeared very rigid, as it did not share significant NOEs between the aforementioned structural segments. MD simulations confirmed the NOE results. The aqueous dissolution profile of both molecules fits well with their minimum energy conformers’ features, which stem from the NOE data; this was nicely demonstrated, especially in the case of compound II.     

NMR studies of molecular conformations in alpha-cyclodextrin

A new approach for analysis of NMR parameters is proposed. The experimental data set includes scalar couplings, NOEs, and residual dipolar couplings. The method, which aims at construction of the conformational distribution function, is applied to alpha-cyclodextrin in isotropic solution and dissolved in a dilute liquid crystal. An attempt to analyze the experimental data using an average molecular conformation resulted in unacceptable errors. Our approach rests on the maximum entropy method (ME), which gives the flattest possible distribution, consistent with the experimental data. Very good agreement between experimental and calculated NMR parameters was observed. In fact, two conformational states were required in order to obtain a satisfactory agreement between calculated and experimental data. In addition, good agreement with Langevin dynamics computer simulations was obtained.     

Conformational Preferences of a β‐Octapeptide as Function of Solvent and Force‐Field Parameters

The ability to design properly folded β‐peptides with specific biological activities requires detailed insight into the relationship between the amino acid sequence and the secondary and/or tertiary structure of the peptide. One of the most frequently used spectroscopic techniques for resolving the structure of a biomolecule is NMR spectroscopy. Because only signal intensities and frequencies are recorded in the experiment, a conformational interpretation of the recorded data is not straightforward, especially for flexible molecules. The occurrence of conformational and/or time averaging, and the limited amount and accuracy of experimental data hamper the precise conformational determination of a biomolecule. In addition, the relation between experimental observables with the underlying conformational ensemble is often only approximately known, thereby aggravating the difficulty of structure determination of biomolecules. The problematic aspects of structure refinement based on NMR nuclear Overhauser effect (NOE) intensities and ³J‐coupling data are illustrated by simulating a β‐octapeptide in explicit MeOH and H₂O as solvents using three different force fields. NMR Data indicated that this peptide would fold into a 3₁₄‐helix in MeOH and into a hairpin in H₂O. Our analysis focused on the conformational space visited by the peptide, on structural properties of the peptide, and on agreement of the MD trajectories with available NMR data. We conclude that 1) although the 3₁₄‐helical structure is present when the peptide is solvated in MeOH, it is not the only relevant conformation, and that 2) the NMR data set available for the peptide, when solvated in H₂O, does not provide sufficient information to derive a single secondary structure, but rather a multitude of folds that fulfill the NOE data set.     

Root mean square deviation probability analysis of molecular dynamics trajectories on DNA

The comparison and detection of the commonalities and differences in multiple structural ensembles is an important step in the use of molecular simulations to gain insight into the conformation and dynamics of complex biomacromolecules. While the average structure is often employed as the representative of an ensemble of structures in such comparisons, dynamic molecular systems with multiple conformational substates call for a more accurate representation that captures the complete dynamical range of the ensemble. We present a probability analysis procedure based on the root-mean-square differences among the structural ensembles that efficiently and accurately performs the relevant comparison.