Enhanced Sampling of Coarse-Grained Transmembrane-Peptide Structure Formation from Hydrogen-Bond Replica Exchange

Protein structure formation in the membrane highlights a grand challenge of sampling in computer simulations, because kinetic traps and slow dynamics make it difficult to find the native state. Exploiting increased fluctuations at higher temperatures can help overcome free-energy barriers, provided the membrane’s structure remains stable. In this work, we apply Hamiltonian replica-exchange molecular dynamics, where we only tune the backbone hydrogen-bond strength to help reduce the propensity of long-lived misfolded states. Using a recently developed coarse-grained model, we illustrate the robustness of the method by folding different WALP transmembrane helical peptides starting from stretched, unstructured conformations. We show the efficiency of the method by comparing to simulations without enhanced sampling, achieving folding in one example after significantly longer simulation times. Analysis of the bilayer structure during folding provides insight into the local membrane deformation during helix formation as a function of chain length (from 16 to 23 residues). Finally, we apply our method to fold the 50-residue-long major pVIII coat protein (fd coat) of the filamentous fd bacteriophage. Our results agree well with experimental structures and atomistic simulations based on implicit membrane models, suggesting that our explicit CG folding protocol can serve as a starting point for better-refined atomistic simulations in a multiscale framework.     

Equilibrium structure and folding of a helix-forming peptide: circular dichroism measurements and replica-exchange molecular dynamics simulations

We have performed experimental measurements and computer simulations of the equilibrium structure and folding of a 21-residue alpha-helical heteropeptide. Far ultraviolet circular dichroism spectroscopy is used to identify the presence of helical structure and to measure the thermal unfolding curve. The observed melting temperature is 296 K, with a folding enthalpy of -11.6 kcal/mol and entropy of -39.6 cal/(mol K). Our simulations involve 45 ns of replica-exchange molecular dynamics of the peptide, using eight replicas at temperatures between 280 and 450 K, and the program CHARMM with a continuum solvent model. In a 30-ns simulation started from a helical structure, conformational equilibrium at all temperatures was reached after 15 ns. This simulation was used to calculate the peptide melting curve, predicting a folding transition with a melting temperature in the 330-350 K range, enthalpy change of -10 kcal/mol, and entropy change of -30 cal/(mol K). The simulation results were also used to analyze the peptide structural fluctuations and the free-energy surface of helix unfolding. In a separate 15-ns replica-exchange molecular dynamics simulation started from the extended structure, the helical conformation was first attained after approximately 2.8 ns, and equilibrium was reached after 10 ns of simulation. These results showed a sequential folding process with a systematic increase in the number of hydrogen bonds until the helical state is reached, and confirmed that the alpha-helical state is the global free-energy minimum for the peptide at low temperatures.     

Solid-State NMR-Restrained Ensemble Dynamics of a Membrane Protein in Explicit Membranes

Solid-state NMR has been used to determine the structures of membrane proteins in native-like lipid bilayer environments. Most structure calculations based on solid-state NMR observables are performed using simulated annealing with restrained molecular dynamics and an energy function, where all nonbonded interactions are represented by a single, purely repulsive term with no contributions from van der Waals attractive, electrostatic, or solvation energy. To our knowledge, this is the first application of an ensemble dynamics technique performed in explicit membranes that uses experimental solid-state NMR observables to obtain the refined structure of a membrane protein together with information about its dynamics and its interactions with lipids. Using the membrane-bound form of the fd coat protein as a model membrane protein and its experimental solid-state NMR data, we performed restrained ensemble dynamics simulations with different ensemble sizes in explicit membranes. For comparison, a molecular dynamics simulation of fd coat protein was also performed without any restraints. The average orientation of each protein helix is similar to a structure determined by traditional single-conformer approaches. However, their variations are limited in the resulting ensemble of structures with one or two replicas, as they are under the strong influence of solid-state NMR restraints. Although highly consistent with all solid-state NMR observables, the ensembles of more than two replicas show larger orientational variations similar to those observed in the molecular dynamics simulation without restraints. In particular, in these explicit membrane simulations, Lys⁴⁰, residing at the C-terminal side of the transmembrane helix, is observed to cause local membrane curvature. Therefore, compared to traditional single-conformer approaches in implicit environments, solid-state NMR restrained ensemble simulations in explicit membranes readily characterize not only protein dynamics but also protein-lipid interactions in detail.     

Ab initio folding simulation of Trpcage by replica exchange with hybrid Hamiltonian

Replica-exchange molecular dynamics simulations with hybrid Hamiltonian in explicit solvent were performed to study the folding of a designed 20-residue miniprotein, Trpcage, from a fully extended structure. During the simulations several folding/unfolding events happened. In the folded states the majority of experimentally observed NMR NOE restraints are satisfied. The folded structures have root mean squared deviation of 2.0 A with respect to the NMR structures considering all heavy atoms. The free-energy surface constructed by the hybrid Hamiltonian simulations is similar to the one built by a standard replica-exchange simulation which started from the native structure. Consistent with previous experimental observation, a pre-existing hydrophobic collapse in the unfolded state is detected by investigating the desolvation behavior of Trpcage. At room temperature, an intermediate state featured by a misfolded core, a nearly formed alpha-helix segment and an absence of 3(10)-helix is found. The replica exchange with hybrid Hamiltonian method is shown here to be capable of resolving the folding picture of the miniprotein.     

Monte Carlo folding of trans-membrane helical peptides in an implicit generalized Born membrane

An efficient Monte Carlo (MC) algorithm using concerted backbone rotations is combined with a recently developed implicit membrane model to simulate the folding of the hydrophobic transmembrane domain M2TM of the M2 protein from influenza A virus and Sarcolipin at atomic resolution. The implicit membrane environment is based on generalized Born theory and has been calibrated against experimental data. The MC sampling has previously been used to fold several small polypeptides and been shown to be equivalent to molecular dynamics (MD). In combination with a replica exchange algorithm, M2TM is found to form continuous membrane spanning helical conformations for low temperature replicas. Sarcolipin is only partially helical, in agreement with the experimental NMR structures in lipid bilayers and detergent micelles. Higher temperature replicas exhibit a rapidly decreasing helicity, in agreement with expected thermodynamic behavior. To exclude the possibility of an erroneous helical bias in the simulations, the model is tested by sampling a synthetic Alanine-rich polypeptide of known helicity. The results demonstrate there is no overstabilization of helical conformations, indicating that the implicit model captures the essential components of the native membrane environment for M2TM and Sarcolipin.     

Structural and orientational constraints of bacteriorhodopsin in purple membranes determined by oriented-sample solid-state NMR spectroscopy

We report for the first time, oriented-sample solid-state NMR experiments, specifically polarization inversion spin exchange at the magic angle (PISEMA) and 1H-15N heteronuclear chemical shift correlation (HETCOR), applied to an integral seven-transmembrane protein, bacteriorhodopsin (bR), in natural membranes. The spectra of [15N]Met-bR revealed clearly distinguishable signals from the helical and loop regions. By deconvolution of the helix resonances, it was possible to establish constraints for some helix tilt angles. It was estimated that the extracellular section of helix B has a tilt of less than 5 degrees from the membrane normal, while the tilt of helix A was estimated to be 18-22 degrees , both of which are in agreement with most crystal structures. Comparison of the experimental PISEMA spectrum with simulated spectra based on crystal structures showed that PISEMA and HETCOR experiments are extremely sensitive to the polytopic protein structure, and the solid-state NMR spectra for membrane-embedded bR matched most favorably with the recent 1FBB electron crystallography structure. These results suggest that this approach has the potential to yield structural and orientational constraints for large integral polytopic proteins whilst intercalated and functionally competent in a natural membrane.     

Blind Test of Physics-Based Prediction of Protein Structures

We report here a multiprotein blind test of a computer method to predict native protein structures based solely on an all-atom physics-based force field. We use the AMBER 96 potential function with an implicit (GB/SA) model of solvation, combined with replica-exchange molecular-dynamics simulations. Coarse conformational sampling is performed using the zipping and assembly method (ZAM), an approach that is designed to mimic the putative physical routes of protein folding. ZAM was applied to the folding of six proteins, from 76 to 112 monomers in length, in CASP7, a community-wide blind test of protein structure prediction. Because these predictions have about the same level of accuracy as typical bioinformatics methods, and do not utilize information from databases of known native structures, this work opens up the possibility of predicting the structures of membrane proteins, synthetic peptides, or other foldable polymers, for which there is little prior knowledge of native structures. This approach may also be useful for predicting physical protein folding routes, non-native conformations, and other physical properties from amino acid sequences.     

Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment

Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives, successfully used for producing proteins for solution NMR studies, is yeast expression systems, particularly Pichia pastoris. We report on successful implementation and optimization of isotope labeling protocols, previously used for soluble secreted proteins, to produce homogeneous samples of a eukaryotic seven-transmembrane helical protein, rhodopsin from Leptosphaeria maculans. Even in shake-flask cultures, yields exceeded 5 mg of purified uniformly ¹³C,¹⁵N-labeled protein per liter of culture. The protein was stable (at least several weeks at 5°C) and functionally active upon reconstitution into lipid membranes at high protein-to-lipid ratio required for solid-state NMR. The samples gave high-resolution ¹³C and ¹⁵N solid-state magic angle spinning NMR spectra, amenable to a detailed structural analysis. We believe that similar protocols can be adopted for challenging mammalian targets, which often resist characterization by other structural methods.     

Computer simulations of de novo designed helical proteins.

In the context of reduced protein models, Monte Carlo simulations of three de novo designed helical proteins (four-member helical bundle) were performed. At low temperatures, for all proteins under consideration, protein-like folds having different topologies were obtained from random starting conformations. These simulations are consistent with experimental evidence indicating that these de novo designed proteins have the features of a molten globule state. The results of Monte Carlo simulations suggest that these molecules adopt four-helix bundle topologies. They also give insight into the possible mechanism of folding and association, which occurs in these simulations by on-site assembly of the helices. The low-temperature conformations of all three sequences have the features of a molten globule state.     

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.     

Helix-coil transition of alanine peptides in water: force field dependence on the folded and unfolded structures

The force fields used in classical modeling studies are semiempirical in nature and rely on their validation by comparison of simulations with experimental data. The all-atom replica-exchange molecular dynamics (REMD) methodology allows us to calculate the thermodynamics of folding/unfolding of peptides and small proteins, and provides a way of evaluating the reliability of force fields. We apply the REMD to obtain equilibrium folding/unfolding thermodynamics of a 21-residue peptide containing only alanine residues in explicit aqueous solution. The thermodynamics of this peptide is modeled with both the OPLS/AA/L and the A94/MOD force fields. We find that the helical content and the values for the helix propagation and nucleation parameters for this alanine peptide are consistent with measurements on similar peptides and with calculations using the modified AMBER force field (A94/MOD). The nature of conformations, both folded and unfolded, that contributes to the helix-coil transition profile, however, is quite different between these two force fields.     

Solid-state NMR studies of the membrane-bound closed state of the colicin E1 channel domain in lipid bilayers.

The colicin E1 channel polypeptide was shown to be organized anisotropically in membranes by solid-state NMR analysis of samples of uniformly 15N-labeled protein in oriented planar phospholipid bilayers. The 190 residue C-terminal colicin E1 channel domain is the largest polypeptide to have been characterized by 15N solid-state NMR spectroscopy in oriented membrane bilayers. The 15N-NMR spectra of the colicin E1 show that: (1) the structure and dynamics are independent of anionic lipid content in both oriented and unoriented samples; (2) assuming the secondary structure of the polypeptide is helical, there are both trans-membrane and in-plane helical segments; (3) trans-membrane helices account for approximately 20-25% of the channel polypeptide, which is equivalent to 38-48 residues of the 190-residue polypeptide. The results of the two-dimensional PISEMA spectrum are interpreted in terms of a single trans-membrane helical hairpin inserted into the bilayer from each channel molecule. These data are also consistent with this helical hairpin being derived from the 38-residue hydrophobic segment near the C-terminus of the colicin E1 channel polypeptide.     

The unfolded state of the villin headpiece helical subdomain: computational studies of the role of locally stabilized structure

The 36 residue villin headpiece helical subdomain (HP36) is one of the fastest cooperatively folding proteins, folding on the microsecond timescale. HP36's simple three helix topology, fast folding and small size have made it an attractive model system for computational and experimental studies of protein folding. Recent experimental studies have explored the denatured state of HP36 using fragment analysis coupled with relatively low-resolution spectroscopic techniques. These studies have shown that there is apparently only a small tendency to form locally stabilized secondary structure. Here, we complement the experimental studies by using replica exchange molecular dynamics with explicit solvent to investigate the structural features of these peptide models of unfolded HP36. To ensure convergence, two sets of simulations for each fragment were performed with different initial structures, and simulations were continued until these generated very similar final ensembles. These simulations reveal low populations of native-like structure and early folding events that cannot be resolved by experiment. For each fragment, calculated J-coupling constants and helical propensities are in good agreement with experimental trends. HP-1, corresponding to residues 41 to 53 and including the first alpha-helix, contains the highest helical population. HP-3, corresponding to residues 62 through 75 and including the third alpha-helix, contains a small population of helical turn residing at the N terminus while HP-2, corresponding to residues 52 through 61 and including the second alpha-helix, formed little to no structure in isolation. Overall, HP-1 was the only fragment to adopt a native-like conformation, but the low population suggests that formation of significant structure only occurs after formation of specific tertiary interactions.     

Solid-state NMR structures of integral membrane proteins

Abstract

Solid-state NMR is unique for its ability to obtain three-dimensional structures and to measure atomic-resolution structural and dynamic information for membrane proteins in native lipid bilayers. An increasing number and complexity of integral membrane protein structures have been determined by solid-state NMR using two main methods. Oriented sample solid-state NMR uses macroscopically aligned lipid bilayers to obtain orientational restraints that define secondary structure and global fold of embedded peptides and proteins and their orientation and topology in lipid bilayers. Magic angle spinning (MAS) solid-state NMR uses unoriented rapidly spinning samples to obtain distance and torsion angle restraints that define tertiary structure and helix packing arrangements. Details of all current protein structures are described, highlighting developments in experimental strategy and other technological advancements. Some structures originate from combining solid- and solution-state NMR information and some have used solid-state NMR to refine X-ray crystal structures. Solid-state NMR has also validated the structures of proteins determined in different membrane mimetics by solution-state NMR and X-ray crystallography and is therefore complementary to other structural biology techniques. By continuing efforts in identifying membrane protein targets and developing expression, isotope labelling and sample preparation strategies, probe technology, NMR experiments, calculation and modelling methods and combination with other techniques, it should be feasible to determine the structures of many more membrane proteins of biological and biomedical importance using solid-state NMR. This will provide three-dimensional structures and atomic-resolution structural information for characterising ligand and drug interactions, dynamics and molecular mechanisms of membrane proteins under physiological lipid bilayer conditions.     

Exploring the energy landscape of protein folding using replica-exchange and conventional molecular dynamics simulations

Two independent replica-exchange molecular dynamics (REMD) simulations with an explicit water model were performed of the Trp-cage mini-protein. In the first REMD simulation, the replicas started from the native conformation, while in the second they started from a nonnative conformation. Initially, the first simulation yielded results qualitatively similar to those of two previously published REMD simulations: the protein appeared to be over-stabilized, with the predicted melting temperature 50-150K higher than the experimental value of 315K. However, as the first REMD simulation progressed, the protein unfolded at all temperatures. In our second REMD simulation, which starts from a nonnative conformation, there was no evidence of significant folding. Transitions from the unfolded to the folded state did not occur on the timescale of these simulations, despite the expected improvement in sampling of REMD over conventional molecular dynamics (MD) simulations. The combined 1.42 micros of simulation time was insufficient for REMD simulations with different starting structures to converge. Conventional MD simulations at a range of temperatures were also performed. In contrast to REMD, the conventional MD simulations provide an estimate of Tm in good agreement with experiment. Furthermore, the conventional MD is a fraction of the cost of REMD and continuous, realistic pathways of the unfolding process at atomic resolution are obtained.     

Molecular Dynamics Simulations Reveal the HIV-1 Vpu Transmembrane Protein to Form Stable Pentamers

The human immunodeficiency virus type I (HIV-1) Vpu protein is 81 residues long and has two cytoplasmic and one transmembrane (TM) helical domains. The TM domain oligomerizes to form a monovalent cation selective ion channel and facilitates viral release from host cells. Exactly how many TM domains oligomerize to form the pore is still not understood, with experimental studies indicating the existence of a variety of oligomerization states. In this study, molecular dynamics (MD) simulations were performed to investigate the propensity of the Vpu TM domain to exist in tetrameric, pentameric, and hexameric forms. Starting with an idealized α-helical representation of the TM domain, a thorough search for the possible orientations of the monomer units within each oligomeric form was carried out using replica-exchange MD simulations in an implicit membrane environment. Extensive simulations in a fully hydrated lipid bilayer environment on representative structures obtained from the above approach showed the pentamer to be the most stable oligomeric state, with interhelical van der Waals interactions being critical for stability of the pentamer. Atomic details of the factors responsible for stable pentamer structures are presented. The structural features of the pentamer models are consistent with existing experimental information on the ion channel activity, existence of a kink around the Ile17, and the location of tetherin binding residues. Ser23 is proposed to play an important role in ion channel activity of Vpu and possibly in virus propagation.     

Comprehensive evaluation of solution nuclear magnetic resonance spectroscopy sample preparation for helical integral membrane proteins

The preparation of high quality samples is a critical challenge for the structural characterization of helical integral membrane proteins. Solving the structures of this diverse class of proteins by solution nuclear magnetic resonance spectroscopy (NMR) requires that well-resolved 2D ¹H/¹⁵N chemical shift correlation spectra be obtained. Acquiring these spectra demands the production of samples with high levels of purity and excellent homogeneity throughout the sample. In addition, high yields of isotopically enriched protein and efficient purification protocols are required. We describe two robust sample preparation methods for preparing high quality, homogeneous samples of helical integral membrane proteins. These sample preparation protocols have been combined with screens for detergents and sample conditions leading to the efficient production of samples suitable for solution NMR spectroscopy. We have examined 18 helical integral membrane proteins, ranging in size from approximately 9 kDa to 29 kDa with 1–4 transmembrane helices, originating from a number of bacterial and viral genomes. 2D ¹H/¹⁵N chemical shift correlation spectra acquired for each protein demonstrate well-resolved resonances, and >90% detection of the predicted resonances. These results indicate that with proper sample preparation, high quality solution NMR spectra of helical integral membrane proteins can be obtained greatly enhancing the probability for structural characterization of these important proteins.     

核磁共振波谱应用于结构生物学的研究进展

综述了核磁共振波谱在结构生物学研究中的进展。在溶液中测定生物大分子的结构,分子大小的限制正被减少,尽管新结构的测定仍然需要付出比较大的努力。核磁共振是一个有效的手段,可用于研究在许多细胞过程中存在的弱的或者瞬态的蛋白质-蛋白质相互作用。结构的柔性在蛋白质分子功能中起了中心作用。由于最近方法学的发展,使NMR可以表征蛋白质的动力学,从而可以对分子机制有新的认识。核磁共振波谱可以在原子分辨率下表征无序的蛋白质系统,可以研究折叠路径。跨膜蛋白在细胞中起了关键作用,这使它们成为药物的靶标。应用液体和固体核磁共振技术已经成功测定了跨膜蛋白质的结构。