Solid-state NMR, electrophysiology and molecular dynamics characterization of human VDAC2.

The voltage-dependent anion channel (VDAC) is the most abundant protein of the outer mitochondrial membrane and constitutes the major pathway for the transport of ADP, ATP, and other metabolites. In this multidisciplinary study we combined solid-state NMR, electrophysiology, and molecular dynamics simulations, to study the structure of the human VDAC isoform 2 in a lipid bilayer environment. We find that the structure of hVDAC2 is similar to the structure of hVDAC1, in line with recent investigations on zfVDAC2. However, hVDAC2 appears to exhibit an increased conformational heterogeneity compared to hVDAC1 which is reflected in broader solid-state NMR spectra and less defined electrophysiological profiles.

What stabilizes the 3(14)-helix in beta3-peptides? A conformational analysis using molecular simulation.

Beta-peptides are analogs of natural alpha-peptides and form a variety of remarkably stable structures. Having an additional carbon atom in the backbone of each residue, their folded conformation is not only influenced by the side-chain sequence but also and foremost by their substitution pattern. The precise mechanism by which the side chains interact with the backbone is, however, hitherto not completely known. To unravel the various effects by which the side chains influence the backbone conformation, we quantify to which extent the dihedral angles of a beta(3)-substituted peptide with an additional methyl group on the central C(alpha)-atom can be regarded as independent degrees of freedom and analyze the distributions of these dihedral angles. We also selectively capture the steric effect of substituents on the C(alpha)- and C(beta)-atoms of the central residue by alchemically changing them into dummy atoms, which have no nonbonded interactions. We find that the folded state of the beta(3)-peptide is primarily stabilized by a steric exclusion of large parts of the unfolded state (entropic effect) and only subsequently by mutual dependence of the psi-dihedral angles (enthalpic effect). The folded state of beta-peptides is stabilized by a different mechanism than that of alpha-peptides.

Structure determination of a flexible cyclic peptide based on NMR and MD simulation 3J-coupling.

Molecular dynamics (MD) simulations, in which experimental values such as nuclear Overhauser effects (NOEs), dipolar couplings, (3)J-coupling constants or crystallographic structure factors are used to bias the values of specific molecular properties towards experimental ones, are often carried out to study the structure refinement of peptides and proteins. However, (3)J-coupling constants are usually not employed because of the multiplicity of torsional angle values (phi) corresponding to each (3)J-coupling constant value. Here, we apply the method of adaptively enforced restraining using a local-elevation (LE) biasing potential energy function in which a memory function penalizes conformations in case both the average <(3)J> and the current (3)J-values deviate from the experimental target value. Then, the molecule is forced to sample other parts of the conformational space, thereby being able to cross high energy barriers and to bring the simulated average <(3)J> close to the measured <(3)J> value. Herein, we show the applicability of this method in structure refinement of a cyclo-beta-tetrapeptide by enforcing the (3)J-value restraining with LE on twelve backbone torsional angles. The resulting structural ensemble satisfies the experimental (3)J-coupling data better than the NMR model structure derived using conventional single-structure refinement based on these data. Thus, application of local-elevation search MD simulation in combination with biasing towards (3)J-coupling makes it possible to use (3)J-couplings quantitatively in structure determination of peptides.

A combined solid-state NMR and MD characterization of the stability and dynamics of the HET-s(218-289) prion in its amyloid conformation.

The three-dimensional structure of amyloid fibrils of the prion-forming part of the HET-s protein [HET-s(218-289)], as determined by solid-state NMR, contains rigid and remarkably well-ordered parts, as witnessed by the narrow solid-state NMR line widths for this system. On the other hand, high-resolution magic-angle-spinning (HRMAS) NMR results have shown that HET-s(218-289) amyloid fibrils contain highly flexible parts as well. Here, we further explore this unexpected behaviour using solid-state NMR and molecular dynamics (MD). The NMR data provide new information on order and dynamics in the rigid and flexible parts of HET-s(218-289), respectively. The MD study addresses whether or not small multimers, in an amyloid conformation, are stable on the 10 ns timescale of the MD run and provides insight into the dynamic parameters on the nanosecond timescale. The atom-positional, root-mean-squared fluctuations (RMSFs) and order parameters S(2) obtained are in agreement with the NMR data. A flexible loop and the N terminus exhibit dynamics on the ps-ns timescale, whereas the hydrophobic core of HET-s(218-289) is rigid. The high degree of order in the core region of HET-s(218-289) amyloids, as observed in the MD simulations, is in agreement with the narrow, solid-state, NMR lines. Finally, we employed MD to predict the behaviour of the salt-bridge network in HET-s(218-289), which cannot be obtained easily by experiment. Simulations at different temperatures indicated that the network is highly dynamic and that it contributes to the thermostability of the HET-s(218-289) amyloids.

Interpreting experimental data by using molecular simulation instead of model building.

A proper description of the conformational equilibrium of polypeptides or proteins is essential for a correct description of their function. The conformational ensembles from 16 molecular dynamic simulations of two beta- heptapeptides were used to interpret the primary NMR data, which were also compared to a set of NMR model structures (see graphic).One of the most used spectroscopic techniques for resolving the structure of a biomolecule, such as a protein or peptide, is NMR spectroscopy. Because only NMR signal intensities and frequencies are measured in the experiment, a conformational interpretation of the primary data, that is, measured data, is not straightforward, especially for flexible molecules. It is hampered by the occurrence of conformational and/or time-averaging, by insufficient number of experimental data and by insufficient accuracy of experimental data. All three problematic aspects of structure refinement based on NMR nuclear Overhauser effect (NOE) intensities and (3)J coupling data are illustrated by using two beta-heptapeptides in methanol as an example. We have performed 16 molecular dynamics (MD) simulations between 20 to 100 ns in length of unrestrained and NOE distance-restrained cases (instantaneous and time-averaged) of two beta-heptapeptides with a central beta-HAla(alpha-OH) amino acid in methanol at two different temperatures using two different GROMOS force-field parameter sets, 45 A3 and 53 A6. The created conformational ensembles were used to interpret the primary NMR data on these molecules. They also were compared to a set of NMR model structures derived by single-structure refinement in vacuo by using standard techniques. It is shown that the conformational interpretation of measured experimental data can be significantly improved by using unrestrained, instantaneous and time-averaged restrained MD simulations of the peptides by using a thermodynamically calibrated force field and by explicitly including solvent degrees of freedom.

Influence of backbone fluorine substitution upon the folding equilibrium of a beta-heptapeptide.

Explicit solvent molecular dynamics (MD) simulations of three beta-heptapeptides with a central beta- HAla(alpha-F) amino acid (Figure 1) in methanol are reported. They aim at an analysis of the conformational consequences of C(alpha) carbon atom bound fluoro atoms, and the particular configuration of the central fluoro-beta-amino acid: peptide 3 with an S configuration of the C(alpha) bound fluor atom, peptide 4 with an R configuration of the C(alpha) bound fluor atom, and peptide 5 with a difluoro substitution at the C(alpha) atom of residue 4. The NMR and CD spectra of these three beta-peptides were earlier (Mathad et al. Helv. Chim. Acta 2005, 88, 266-280) interpreted to indicate a decrease in propensity of 3(14)-helical structure from peptide 4 to peptide 5 to peptide 3. This result was at odds with previous experimental data for beta-heptapeptides with a central beta-HAla(alpha-Me) amino acid which showed that the beta-heptapeptide with the S,S configuration of the central beta-HAla(alpha-Me) was the most 3(14)-helical, whereas the S,R configuration did not lead to any detected helicity. The reported MD simulations resolve this paradox. The MD trajectories of all three peptides do agree with the primary, measured data: NMR nuclear Overhauser effect (NOE) atom-atom distance bounds and (3)J-coupling constants. A conformational analysis of the MD trajectory conformations shows, however, a decrease in 3(14)-helical character from peptide 3 to peptide 5 to peptide 4, which is in line with the results for the nonfluorinated peptides. It is shown that interpretation of NMR NOE data using single-structure refinement in vacuo based on local (along the sequence) and limited atom-atom distance data as in ref 1 (Mathad et al. Helv. Chim. Acta 2005, 88, 266-280) may lead to molecular structures that are not representative for the ensemble of molecular conformations.

A molecular dynamics study of the ASC and NALP1 pyrin domains at neutral and low pH.

The pyrin domain is one of four subfamilies of the death domain superfamily of proteins, all members of which share a similar three-dimensional fold with a structure comprising five or six antiparallel alpha-helices. The pyrin domain of the ASC (six-helical fold) and of the NALP1 (five-helical fold) proteins were simulated at two different pH values, 3.7 and 6.5, with two different force-field parameter sets, and the molecular dynamics simulation trajectories were compared to NMR experimental data. The two force fields that were used did not show very different results. The simulations of NALP1 at pH 6.5 largely satisfied the experimental NOE atom-atom distance bounds that were measured at pH 6.5, and preserved its tertiary structure. The simulations at pH 3.7 showed a denaturation of the protein. The simulations of ASC at pH 3.7 only satisfied the experimental NOE atom-atom distance bounds that were measured at pH 3.7 if either three acidic side chains (Asp48, Glu64 and Asp75) or only two (Glu64 and Asp75) were not protonated. This indicates that the ASC tertiary structure is stabilized by salt bridges at low pH. A corresponding analysis for NALP1 at pH 3.7 only yielded one possible salt bridge, but this did not stabilize the tertiary structure at low pH. The results show that the particular protonation states of acidic side chains in the protein interior might be crucial to properly modeling these proteins at low pH.

NMR-based detection of hydrogen/deuterium exchange in liposome-embedded membrane proteins.

Membrane proteins play key roles in biology. Determination of their structure in a membrane environment, however, is highly challenging. To address this challenge, we developed an approach that couples hydrogen/deuterium exchange of membrane proteins to rapid unfolding and detection by solution-state NMR spectroscopy. We show that the method allows analysis of the solvent protection of single residues in liposome-embedded proteins such as the 349-residue Tom40, the major protein translocation pore in the outer mitochondrial membrane, which has resisted structural analysis for many years.

ß-Barrel mobility underlies closure of the voltage-dependent anion channel.

The voltage-dependent anion channel (VDAC) is the major protein in the outer mitochondrial membrane, where it mediates transport of ATP and ADP. Changes in its permeability, induced by voltage or apoptosis-related proteins, have been implicated in apoptotic pathways. The three-dimensional structure of VDAC has recently been determined as a 19-stranded ß-barrel with an in-lying N-terminal helix. However, its gating mechanism is still unclear. Using solid-state NMR spectroscopy, molecular dynamics simulations, and electrophysiology, we show that deletion of the rigid N-terminal helix sharply increases overall motion in VDAC's ß-barrel, resulting in elliptic, semicollapsed barrel shapes. These states quantitatively reproduce conductance and selectivity of the closed VDAC conformation. Mutation of the N-terminal helix leads to a phenotype intermediate to the open and closed states. These data suggest that the N-terminal helix controls entry into elliptic ß-barrel states which underlie VDAC closure. Our results also indicate that ß-barrel channels are intrinsically flexible.

Structure and dynamics of two beta-peptides in solution from molecular dynamics simulations validated against experiment.

We have studied two different beta-peptides in methanol using explicit solvent molecular dynamics simulations and the GROMOS 53A6 force field: a heptapeptide (peptide 1) expected to form a left-handed 3(14)-helix, and a hexapeptide (peptide 2) expected to form a beta-hairpin in solution. Our analysis has focused on identifying and analyzing the stability of the dominant secondary structure conformations adopted by the peptides, as well as on comparing the experimental NOE distance upper bounds and 3J-coupling values with their counterparts calculated on the basis of the simulated ensembles. Moreover, we have critically compared the present results with the analogous results obtained with the GROMOS 45A3 (peptide 1) and 43A1 (peptide 2) force fields. We conclude that within the limits of conformational sampling employed here, the GROMOS 53A6 force field satisfactorily reproduces experimental findings regarding the behavior of short beta-peptides, with accuracy that is comparable to but not exceeding that of the previous versions of the force field. GCE legend Conformational clustering analysis of the simulated ensemble of a ss-hexapeptide with two different simulation setups (a and b). The central members of all of the clusters populating more than 5% of all of the structures are shown, together with the most dominant hydrogen bonds and the corresponding percentages of cluster members containing them.

Atomic models of de novo designed cc beta-Met amyloid-like fibrils.

The common characteristics of amyloid and amyloid-like fibrils from disease- and non-disease-associated proteins offer the prospect that well-defined model systems can be used to systematically dissect the driving forces of amyloid formation. We recently reported the de novo designed cc beta peptide model system that forms a native-like coiled-coil structure at low temperatures and which can be switched to amyloid-like fibrils by increasing the temperature. Here, we report a detailed molecular description of the system in its fibrillar state by characterizing the cc beta-Met variant using several microscopic techniques, circular dichroism spectroscopy, X-ray fiber diffraction, solid-state nuclear magnetic resonance, and molecular dynamics calculations. We show that cc beta-Met forms amyloid-like fibrils of different morphologies on both the macroscopic and atomic levels, which can be controlled by variations of assembly conditions. Interestingly, heterogeneity is also observed along single fibrils. We propose atomic models of the cc beta-Met amyloid-like fibril, which are in good agreement with all experimental data. The models provide a rational explanation why oxidation of methionine residues completely abolishes cc beta-Met amyloid fibril formation, indicating that a small number of site-specific hydrophobic interactions can play a major role in the packing of polypeptide-chain segments within amyloid fibrils. The detailed structural information available for the cc beta model system provides a strong molecular basis for understanding the influence and relative contribution of hydrophobic interactions on native-state stability, kinetics of fibril formation, fibril packing, and polymorphism.

Simulation of beta-depsipeptides: the effect of missing hydrogen-bond donors on their folding equilibria.

beta-Depsipeptides are beta-peptides in which one or more peptide linkages are replaced by ester linkages, resulting in a loss of a hydrogen-bond donor (N--H) and weakening of the corresponding carbonyl hydrogen-bond acceptor moiety. The effects of three of such peptide by ester substitutions in a hepta-beta-peptide upon its (un)folding equilibrium in methanol solution are investigated using molecular dynamics simulations and compared to experimental data from NMR spectroscopy. The simulated conformational ensembles largely reproduce the experimentally measured NOE and 3J-coupling constant data for the three different hepta-beta-peptides, and confirm the relative stabilities of the 3(14)-helical conformation, which is most weakened by substitution of the 4th peptide linkage and least by substitution of the 6th peptide linkage. The simulations are complementary to the experimental data by providing detailed insight into the conformational distributions that are compatible with the experimentally measured average values of observables.