Full Reconstruction of a Vectorial Protein Folding Pathway by Atomic Force Microscopy and Molecular Dynamics Simulations

During co-translational folding, the nascent polypeptide chain is extruded sequentially from the ribosome exit tunnel and, under severe conformational constraints, is dictated by its one-dimensional geometry. How do such vectorial constraints impact the folding pathway? Here, we combine single-molecule atomic force spectroscopy and steered molecular dynamics simulations to examine protein folding in the presence of one-dimensional constraints that are similar to those imposed on the nascent polypeptide chain. The simulations exquisitely reproduced the experimental unfolding and refolding force extension relationships and led to the full reconstruction of the vectorial folding pathway of a large polypeptide, the 253-residue consensus ankyrin repeat protein, NI6C. We show that fully stretched and then relaxed NI6C starts folding by the formation of local secondary structures, followed by the nucleation of three N-terminal repeats. This rate-limiting step is then followed by the vectorial and sequential folding of the remaining repeats. However, after partial unfolding, when allowed to refold, the C-terminal repeats successively regain structures without any nucleation step by using the intact N-terminal repeats as a template. These results suggest a pathway for the co-translational folding of repeat proteins and have implications for mechanotransduction.     

Glycosylation Effects on FSH-FSHR Interaction Dynamics: A Case Study of Different FSH Glycoforms by Molecular Dynamics Simulations

The gonadotropin known as follicle-stimulating hormone (FSH) plays a key role in regulating reproductive processes. Physiologically active FSH is a glycoprotein that can accommodate glycans on up to four asparagine residues, including two sites in the FSHα subunit that are critical for biochemical function, plus two sites in the β subunit, whose differential glycosylation states appear to correspond to physiologically distinct functions. Some degree of FSHβ hypo-glycosylation seems to confer advantages toward reproductive fertility of child-bearing females. In order to identify possible mechanistic underpinnings for this physiological difference we have pursued computationally intensive molecular dynamics simulations on complexes between the high affinity site of the gonadal FSH receptor (FSHR) and several FSH glycoforms including fully-glycosylated (FSH²⁴), hypo-glycosylated (e.g., FSH¹⁵), and completely deglycosylated FSH (dgFSH). These simulations suggest that deviations in FSH/FSHR binding profile as a function of glycosylation state are modest when FSH is adorned with only small glycans, such as single N-acetylglucosamine residues. However, substantial qualitative differences emerge between FSH¹⁵ and FSH²⁴ when FSH is decorated with a much larger, tetra-antennary glycan. Specifically, the FSHR complex with hypo-glycosylated FSH¹⁵ is observed to undergo a significant conformational shift after 5–10 ns of simulation, indicating that FSH¹⁵ has greater conformational flexibility than FSH²⁴ which may explain the more favorable FSH¹⁵ kinetic profile. FSH¹⁵ also exhibits a stronger binding free energy, due in large part to formation of closer and more persistent salt-bridges with FSHR.     

The Structure of Liquid Methanol: A Molecular Dynamics Study Using Three-Site Models

Abstract

The structure of liquid methanol at 298.15 K is investigated by performing molecular dynamics (MD) simulations in NVE ensemble using two 3-site force field models. The simulated structural results are compared with the recent neutron diffraction (ND) results obtained at the partial pair distribution function (pdf) level by employing H/D substitution on the hydroxyl hydrogen, Ho. Overall agreement is found between the simulated and experimental total intermolecular radial distribution functions (rdfs). The ability of the 3-site model simulations to satisfactorily reproduce experimental X—X (X = C, O or H- a methyl hydrogen) intermolecular partial distribution function, dominated by contributions from the methyl group. demonstrates that the methyl group does not participate in any bonding in the liquid. However, a comparison between the simulated and experimental Ho—Ho and X—Ho functions reveals that discrepancies still exist at a quantitative level.     

Secondary Structure Propensities in Peptide Folding Simulations: A Systematic Comparison of Molecular Mechanics Interaction Schemes

We present a systematic study directed toward the secondary structure propensity and sampling behavior in peptide folding simulations with eight different molecular dynamics force-field variants in explicit solvent. We report on the combinational result of force field, water model, and electrostatic interaction schemes and compare to available experimental characterization of five studied model peptides in terms of reproduced structure and dynamics. The total simulation time exceeded 18 μs and included simulations that started from both folded and extended conformations. Despite remaining sampling issues, a number of distinct trends in the folding behavior of the peptides emerged. Pronounced differences in the propensity of finding prominent secondary structure motifs in the different applied force fields suggest that problems point in particular to the balance of the relative stabilities of helical and extended conformations.     

A Kinetic Model of Trp-Cage Folding from Multiple Biased Molecular Dynamics Simulations

Trp-cage is a designed 20-residue polypeptide that, in spite of its size, shares several features with larger globular proteins. Although the system has been intensively investigated experimentally and theoretically, its folding mechanism is not yet fully understood. Indeed, some experiments suggest a two-state behavior, while others point to the presence of intermediates. In this work we show that the results of a bias-exchange metadynamics simulation can be used for constructing a detailed thermodynamic and kinetic model of the system. The model, although constructed from a biased simulation, has a quality similar to those extracted from the analysis of long unbiased molecular dynamics trajectories. This is demonstrated by a careful benchmark of the approach on a smaller system, the solvated Ace-Ala₃-Nme peptide. For the Trp-cage folding, the model predicts that the relaxation time of 3100 ns observed experimentally is due to the presence of a compact molten globule-like conformation. This state has an occupancy of only 3% at 300 K, but acts as a kinetic trap. Instead, non-compact structures relax to the folded state on the sub-microsecond timescale. The model also predicts the presence of a state at of 4.4 Å from the NMR structure in which the Trp strongly interacts with Pro12. This state can explain the abnormal temperature dependence of the and chemical shifts. The structures of the two most stable misfolded intermediates are in agreement with NMR experiments on the unfolded protein. Our work shows that, using biased molecular dynamics trajectories, it is possible to construct a model describing in detail the Trp-cage folding kinetics and thermodynamics in agreement with experimental data.     

基于温度副本交换分子动力学模拟方法研究温度致H1小肽结构变化特征

采用GROMOS43A1力场,用温度副本交换分子动力学模拟方法研究水溶液中H1小肽在4个不同温度下的结构特征.选择H1小肽的初始构象分别为α螺旋和β折叠片,完成了两组独立的36个温度副本交换的分子动力学模拟,一组从α螺旋出发的模拟用来对该小肽的结构特征进行研究,另一组从β折叠片出发的模拟用于验证构象采样的收敛性,每个副本的模拟时间为300ns,共计模拟时间长达21.6μs.在此基础上,研究了H1小肽在温度300K、330K、350K和370K下的结构特征,分析了其主链二面角分布、天然氢键数、β转角的形成概率以及不同温度下偏好采样构象的变化特征等.模拟结果表明,在4个不同温度下,均能够采样到同β折叠片结构的Cα原子均方根偏差最小为0.05nm的构象类,该构象类在4个不同温度300K、330K、350K和370K下分别包含了全部构象的39%、23%、13%和11%.GROMOS43A1力场在刻画小肽的结构方面具有一定的精度,但是在描述氢键方面仍需要加强,H1小肽在不同温度下结构特征的比较能够为分子力场的优化提供重要的帮助.     

Multiplexed-Replica Exchange Molecular Dynamics Method for Protein Folding Simulation

Simulating protein folding thermodynamics starting purely from a protein sequence is a grand challenge of computational biology. Here, we present an algorithm to calculate a canonical distribution from molecular dynamics simulation of protein folding. This algorithm is based on the replica exchange method where the kinetic trapping problem is overcome by exchanging noninteracting replicas simulated at different temperatures. Our algorithm uses multiplexed-replicas with a number of independent molecular dynamics runs at each temperature. Exchanges of configurations between these multiplexed-replicas are also tried, rendering the algorithm applicable to large-scale distributed computing (i.e., highly heterogeneous parallel computers with processors having different computational power). We demonstrate the enhanced sampling of this algorithm by simulating the folding thermodynamics of a 23 amino acid miniprotein. We show that better convergence is achieved compared to constant temperature molecular dynamics simulation, with an efficient scaling to large number of computer processors. Indeed, this enhanced sampling results in (to our knowledge) the first example of a replica exchange algorithm that samples a folded structure starting from a completely unfolded state.     

Conformational Flexibility of the Peptide Hormone Ghrelin in Solution and Lipid Membrane Bound: A Molecular Dynamics Study

Abstract

Human ghrelin is a peptide hormone of 28 aminoacid residues, in which the Ser3 is modified by an octanoyl group. Ghrelin has a major role in the energy metabolism of the human body stimulating growth hormone release as well as food intake. Here we perform molecular dynamics simulations in explicit water and in a DMPC-lipid bilayer/water system in order to structurally characterize this highly flexible peptide and its lipid binding properties. We find a loop structure with residues Glu17 to Lys 20 in the bending region and a short α-helix from residues Pro7 to Glu13. The presence of a lipid membrane does not influence these structural features, but reduces the overall flexibility of the molecule as revealed by reduced root mean square fluctuations of the atom coordinates. The octanoyl-side chain does not insert into the lipid membrane but points into the water phase. The peptide binds to the lipid membrane with its bending region involving residues Arg15, Lys16, Glu17, and Ser18. The implications of these results for the binding pocket of the ghrelin receptor are discussed.     

Conformational Analysis of a Synthetic Antimicrobial Peptide in Water and Membrane-Mimicking Solvents: A Molecular Dynamics Simulation Study

We have investigated structural and dynamic properties of the synthetic peptide hlF1-11 (GRRRSVQWCA, i.e., the first 11 N-terminal amino acids of the human lactoferrin protein) in water, 250 mM NaCl solution, 50% (V/V) water–trifluoroethanol mixture, and in the membrane mimetic 4:4:1 methanol–chloroform–water mixture. For comparison, we have also performed analogous simulations for the biologically inactive control peptide featuring Ala substitutions in the 2, 3, 6 and 9 positions of the hlF1-11 sequence. Statistical analyses of the trajectories indicate that only in the membrane-mimicking medium hlF1-11 adopts preferentially a conformation suitable to interact effectively with the membrane. In this conformation the peptide cationic region is rather flexible and elongated, while the C-terminal hydrophobic moiety appears as a more rigid hairpin-shaped loop approximately perpendicular to the cationic region. No such conformation is statistically relevant for the control peptide.     

Probing Position-Dependent Diffusion in Folding Reactions Using Single-Molecule Force Spectroscopy

Folding of proteins and nucleic acids involves a diffusive search over a multidimensional conformational energy landscape for the minimal-energy structure. When examining the projection of conformational motions onto a one-dimensional reaction coordinate, as done in most experiments, the diffusion coefficient D is generally position dependent. However, it has proven challenging to measure such position-dependence experimentally. We investigated the position-dependence of D in the folding of DNA hairpins as a simple model system in two ways: first, by analyzing the round-trip time to return to a given extension in constant-force extension trajectories measured by force spectroscopy, and second, by analyzing the fall time required to reach a given extension in force jump measurements. These methods yielded conflicting results: the fall time implied a fairly constant D, but the round-trip time implied variations of over an order of magnitude. Comparison of experiments with computational simulations revealed that both methods were strongly affected by experimental artifacts inherent to force spectroscopy measurements, which obscured the intrinsic position-dependence of D. Lastly, we applied Kramers’s theory to the kinetics of hairpins with energy barriers located at different positions along the hairpin stem, as a crude probe of D at different stem positions, and we found that D did not vary much as the barrier was moved along the reaction coordinate. This work underlines the difficulties faced when trying to deduce position-dependent diffusion coefficients from experimental folding trajectories.     

Molecular Dynamics Simulation of Protein Folding with Supersecondary Structure Constraints

We integrate molecular dynamics simulation methods with a newly developed supersecondary structure prediction method and compute the structure of a protein molecule, crambin. The computed structure is similar to the crystal structure with an rms error of 3.94 Å.     

Simulation Performance of a Non-Equilibrium Molecular Dynamics Method Using Density Difference as Driving Force

Abstract

The simulation performance of two NEMD algorithms, the constant density difference (DD) and the constant chemical potential difference (CPD) methods, has been compared in fluctuations of molar flux for He and CH₄ permeation across the ZSM-5 membrane. The CPD method and the DD method are found to give almost the same performance; however, the former seems slightly superior to the latter in terms of low fluctuations of molar flux though the former needs more CPU time than the latter. An advantage of the DD method is that it can simulate the mixed-gas permeation through a membrane under the specification of high and low pressures and the composition of feed gas. It is shown that the density profile of permeating gas could provide important information about the relative resistance at the entrance, inside, and exit regions for permeation.     

Molecular Dynamics Study of Structure,Folding, and Aggregation of Poly-PR and Poly-GR Proteins

Poly-proline-arginine (poly-PR) and poly-glycine-arginine (poly-GR) proteins are believed to be the most toxic dipeptide repeat (DPR) proteins that are expressed by the hexanucleotide repeat expansion mutation in C9ORF72, which are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) diseases. Their structural information and mechanisms of toxicity remain incomplete, however. Using molecular dynamics simulation and all-atom model of proteins, we study folding and aggregation of both poly-PR and poly-GR. The results indicate formation of double-helix structure during the aggregation of poly-PR into dimers, whereas no stable aggregate is formed during the aggregation of poly-GR; the latter only folds into α-helix and double-helix structures that are similar to those formed in the folding of poly-glycine-alanine (poly-GA) protein. Our findings are consistent with the experimental data indicating that poly-PR and poly-GR are less likely to aggregate because of the hydrophilic arginine residues within their structures. Such characteristics could, however, in some respect facilitate migration of the DPR proteins between and within cells and, at the same time, give proline residues the benefits of activating the receptors that regulate ionotropic effect in neurons, resulting in death or malfunction of neurons because of the abnormal increase or decrease of the ion transmission. This may explain the neurotoxicities of poly-PR and poly-GR associated with many neurodegenerative diseases. To our knowledge, this is the first molecular dynamics simulation of the phenomena involving poly-PR and poly-GR proteins.     

Early Relaxation Dynamics in the LC 13 T Cell Receptor in Reaction to 172 Altered Peptide Ligands: A Molecular Dynamics Simulation Study

The interaction between the T cell receptor and the major histocompatibility complex is one of the most important events in adaptive immunology. Although several different models for the activation process of the T cell via the T cell receptor have been proposed, it could not be shown that a structural mechanism, which discriminates between peptides of different immunogenicity levels, exists within the T cell receptor. In this study, we performed systematic molecular dynamics simulations of 172 closely related altered peptide ligands in the same T cell receptor/major histocompatibility complex system. Statistical evaluations yielded significant differences in the initial relaxation process between sets of peptides at four different immunogenicity levels.     

Peptide Aggregation and Pore Formation in a Lipid Bilayer: A Combined Coarse-Grained and All Atom Molecular Dynamics Study

We present a simulation study where different resolutions, namely coarse-grained (CG) and all-atom (AA) molecular dynamics simulations, are used sequentially to combine the long timescale reachable by CG simulations with the high resolution of AA simulations, to describe the complete processes of peptide aggregation and pore formation by alamethicin peptides in a hydrated lipid bilayer. In the 1-μs CG simulations the peptides spontaneously aggregate in the lipid bilayer and exhibit occasional transitions between the membrane-spanning and the surface-bound configurations. One of the CG systems at t = 1 μs is reverted to an AA representation and subjected to AA simulation for 50 ns, during which water molecules penetrate the lipid bilayer through interactions with the peptide aggregates, and the membrane starts leaking water. During the AA simulation significant deviations from the α-helical structure of the peptides are observed, however, the size and arrangement of the clusters are not affected within the studied time frame. Solid-state NMR experiments designed to match closely the setup used in the molecular dynamics simulations provide strong support for our finding that alamethicin peptides adopt a diverse set of configurations in a lipid bilayer, which is in sharp contrast to the prevailing view of alamethicin oligomers formed by perfectly aligned helical alamethicin peptides in a lipid bilayer.     

Structural Study of Cell Attachment Peptide Derived from Laminin by Molecular Dynamics Simulation

Peptides with cell attachment activity are beneficial component of biomaterials for tissue engineering. Conformational structure is one of the important factors for the biological activities. The EF1 peptide (DYATLQLQEGRLHFMFDLG) derived from laminin promotes cell spreading and cell attachment activity mediated by α2β1 integrin. Although the sequence of the EF2 peptide (DFATVQLRNGFPYFSYDLG) is homologous sequence to that of EF1, EF2 does not promote cell attachment activity. To determine whether there are structural differences between EF1 and EF2, we performed replica exchange molecular dynamics (REMD) simulations and conventional molecular dynamics (MD) simulations. We found that EF1 and EF2 had β-sheet structure as a secondary structure around the global minimum. However, EF2 had variety of structures around the global minimum compared with EF1 and has easily escaped from the bottom of free energy. The structural fluctuation of the EF1 is smaller than that of the EF2. The structural variation of EF2 is related to these differences in the structural fluctuation and the number of the hydrogen bonds (H-bonds). From the analysis of H-bonds in the β-sheet, the number of H-bonds in EF1 is larger than that in EF2 in the time scale of the conventional MD simulation, suggesting that the formation of H-bonds is related to the differences in the structural fluctuation between EF1 and EF2. From the analysis of other non-covalent interactions in the amino acid sequences of EF1 and EF2, EF1 has three pairs of residues with hydrophobic interaction, and EF2 has two pairs. These results indicate that several non-covalent interactions are important for structural stabilization. Consequently, the structure of EF1 is stabilized by H-bonds and pairs of hydrophobic amino acids in the terminals. Hence, we propose that non-covalent interactions around N-terminal and C-terminal of the peptides are crucial for maintaining the β-sheet structure of the peptides.     

Molecular Determinants of Epidermal Growth Factor Binding: A Molecular Dynamics Study

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF ligands or the receptor itself.     

Study on Photoresist Transfer in Rotational Near-Field Photolithography Using Molecular Dynamics Simulation

Rotational near-field photolithography (RNFP) has a great potential for nanostructure fabrication. However, photoresist may be transferred between head and disk resulting in failure of the photolithography process. Two models of photoresist transfer from a rotating disk to a photolithography head were developed using molecular dynamics simulation. The simulation results revealed that photoresist transfer is mainly caused by the strong attraction between the hydrogen atoms in the photoresist and the chromium of the head. When the distance between disk and head is less than the critical flying height, the transfer amount increases with the decrease of rotational speed and flying height. Maintaining the flying height above the critical value is the most effective way to ensure the non-transfer of photoresist.

     

Interaction of the Antimicrobial Peptide Polymyxin B1 with Both Membranes of E. coli: A Molecular Dynamics Study

Antimicrobial peptides are small, cationic proteins that can induce lysis of bacterial cells through interaction with their membranes. Different mechanisms for cell lysis have been proposed, but these models tend to neglect the role of the chemical composition of the membrane, which differs between bacterial species and can be heterogeneous even within a single cell. Moreover, the cell envelope of Gram-negative bacteria such as E. coli contains two membranes with differing compositions. To this end, we report the first molecular dynamics simulation study of the interaction of the antimicrobial peptide, polymyxin B1 with complex models of both the inner and outer membranes of E. coli. The results of >16 microseconds of simulation predict that polymyxin B1 is likely to interact with the membranes via distinct mechanisms. The lipopeptides aggregate in the lipopolysaccharide headgroup region of the outer membrane with limited tendency for insertion within the lipid A tails. In contrast, the lipopeptides readily insert into the inner membrane core, and the concomitant increased hydration may be responsible for bilayer destabilization and antimicrobial function. Given the urgent need to develop novel, potent antibiotics, the results presented here reveal key mechanistic details that may be exploited for future rational drug development.