Mechanism Of Membrane Insertion Research Articles

Interactions between Lipid Vesicle Membranes and Single Amino Acid Fibrils: Probable Origin of Specific Neurological Disorders.

Amyloid fibrils are known to be responsible for several neurological disorders, like Alzheimer's disease (AD), Parkinson's disease (PD), etc. For decades, mostly proteins and peptide-based amyloid fibrils have been focused on, and the topic has acknowledged the rise, development, understanding of, and controversy, as well. However, the single amino acid based amyloid fibrils, responsible for several disorders, such as phenylketonuria, tyrosenimia type II, hypermethioninemia, etc., have gotten scientific attention lately. To understand the molecular level pathogenesis of such disorders originated due to the accumulation of single amino acid-based amyloid fibrils, interaction of these fibrils with phospholipid vesicle membranes is found to be an excellent cell-free in vitro setup. Based on such an in vitro setup, these fibrils show a generic mechanism of membrane insertion driven by electrostatic and hydrophobic effects inside the membrane that reduces the integral rigidity of the membrane. Alteration of such fundamental properties of the membrane, therefore, might be referred to as one of the prime pathological factors for the development of these neurological disorders. Hence, such interactions must be investigated in cellular and intracellular compartments to design suitable therapeutic modulators against fibrils.

Perforin-2 clockwise hand-over-hand pre-pore to pore transition mechanism

Perforin-2 (PFN2, MPEG1) is a pore-forming protein that acts as a first line of defense in the mammalian immune system, rapidly killing engulfed microbes within the phagolysosome in macrophages. PFN2 self-assembles into hexadecameric pre-pore rings that transition upon acidification into pores damaging target cell membranes. Here, using high-speed atomic force microscopy (HS-AFM) imaging and line-scanning and molecular dynamics simulation, we elucidate PFN2 pre-pore to pore transition pathways and dynamics. Upon acidification, the pre-pore rings (pre-pore-I) display frequent, 1.8 s−1, ring-opening dynamics that eventually, 0.2 s−1, initiate transition into an intermediate, short-lived, ~75 ms, pre-pore-II state, inducing a clockwise pre-pore-I to pre-pore-II propagation. Concomitantly, the first pre-pore-II subunit, undergoes a major conformational change to the pore state that propagates also clockwise at a rate ~15 s−1. Thus, the pre-pore to pore transition is a clockwise hand-over-hand mechanism that is accomplished within ~1.3 s. Our findings suggest a clockwise mechanism of membrane insertion that with variations may be general for the MACPF/CDC superfamily.

A Zn2+-triggered two-step mechanism of CLIC1 membrane insertion and activation into chloride channels.

ABSTRACTThe chloride intracellular channel (CLIC) protein family displays the unique feature of altering its structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, is found in the cytoplasm or in internal and plasma membranes, with membrane relocalisation linked to endothelial disfunction, tumour proliferation and metastasis. The molecular switch promoting CLIC1 activation remains under investigation. Here, cellular Cl− efflux assays and immunofluorescence microscopy studies have identified intracellular Zn2+ release as the trigger for CLIC1 activation and membrane insertion. Biophysical assays confirmed specific binding to Zn2+, inducing membrane association and enhancing Cl− efflux in a pH-dependent manner. Together, our results identify a two-step mechanism with Zn2+ binding as the molecular switch promoting CLIC1 membrane insertion, followed by pH-mediated activation of Cl− efflux.

Membrane insertion mechanism of the caveola coat protein Cavin1

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

Mechanism of membrane insertion

Virology Human papilloma viruses (HPVs), which are the source of up to 5% of human cancers, use a specialized cellular protein–trafficking complex called retromer to enter the cell from endosomes and transit to the nucleus. Xie et al. previously found that cell-penetrating peptides facilitated movement of part of the capsid protein of the virus into the endosomal membranes. Now, they have isolated multiple “traptamers” that inhibit HPV entry, each at different steps in the entry process. This discovery permitted an elucidation of the sequential steps required for viral entry. Sci. Adv. 10.1126/sciadv.abh4276 (2021).

Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the “fusion peptide” (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.

Elucidating the Mechanism of Membrane Destabilization by the Preferred Modes of Insertion of the Sars-Cov2 Fusion Peptide

Cell penetration of the SARS-Cov2 virus after recognition by the ACE2 receptor requires the fusion of the viral envelope membrane with cellular membranes. The Spike protein (S) of the virus harbors a region identified as the “fusion peptide” (FP) which is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor binding domain of the Spike. Studies have shown that the SARS-Cov2-FP binds Ca2+ ions and perturbs membranes in a calcium-dependent fashion. But the mechanisms of membrane insertion and destabilization remained unclear. We have identified the preferred modes of SARS-Cov2-FP insertion and the role of Ca2+ ions in mediating peptide-membrane interactions from extensive atomistic molecular dynamics (MD) simulations and trajectory analyses. Multiple Ca2+ binding modes in which the ion engaged with different pairs of acidic residues on the FP were observed. A systematic sampling of the interactions of these Ca2+-bound peptide models with lipid membranes showed that SARS-Cov2-FP penetrated the bilayer only in two modes involving different structural domains. In the first mode the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. The second, and more prevalent mode of penetration involves residues L822/F823 in the freed N-terminus of the peptide. The latter insertion was related to the specific mode of Ca2+ association with the peptide in which one Ca2+ is bound to the D830/D839 pair and another to E819/D820. These findings explain the mechanistic role of the necessary cleavage (termed S2’) that frees the N-terminus of the peptide to effectuate the cell-entry of SARS-Cov2. Computational resources provided by the COVID-19 HPC Consortium are gratefully acknowledged.

Stepwise Insertion of Cobra Cardiotoxin CT2 into a Lipid Bilayer Occurs as an Interplay of Protein and Membrane "Dynamic Molecular Portraits".

For many peripheral membrane-binding polypeptides(MBPs), especially β-structural ones, the precise molecular mechanisms of membrane insertion remain unclear. In most cases, only the terminal water-soluble and membrane-bound states have been elucidated, whereas potential functionally important intermediate stages are still not understood in sufficient detail. In this study, we present one of the first successful attempts to describe step-by-step embedding of the MBP cardiotoxin 2 (CT2) from cobra Naja oxiana venom into a lipid bilayer at the atomistic level. CT2 possesses a highly conservative and rigid β-structured three-finger fold shared by many other exogenous and endogenous proteins performing a wide variety of functions. The incorporation of CT2 into the lipid bilayer was analyzed via a 2 μs all-atom molecular dynamics (MD) simulation without restraints. This process was shown to occur over a number of distinct steps, while the geometry of initial membrane attachment drastically differs from that of the final equilibrated state. In the latter one, the hydrophobic platform ("bottom") formed by the tips of the three loops is deeply buried into the lipid bilayer. This agrees well with the NMR data obtained earlier for CT2 in detergent micelles. However, the bottom is too bulky to insert itself into the membrane at once. Instead, the gradual immersion of CT2 initiated by the loop-1 was observed. This initial binding stage was also demonstrated in a series of MD runs with varying starting orientations of the toxin with respect to the bilayer surface. Apart from the nonspecific long-range electrostatic attraction and hydrophobic match/mismatch factor, several specific lipid-binding sites were identified in CT2. They were shown to promote membrane insertion by engaging in strong interactions with lipid head groups, fine-tuning the toxin-membrane accommodation. We therefore propose that the toxin insertion relies on the interplay of nonspecific and specific interactions, which are determined by the "dynamic molecular portraits" of the two players, the protein and the membrane. The proposed model does not require protein oligomerization for membrane insertion and can be further employed to design MBPs with predetermined properties with regard to particular membrane targets.

Membrane insertion mechanism and molecular assembly of the bacteriophage lysis toxin ΦX174-E.

The bacteriophage ΦX174 causes large pore formation in Escherichiacoli and related bacteria. Lysis is mediated by the small membrane-bound toxin ΦX174-E, which is composed of a transmembrane domain and a soluble domain. The toxin requires activation by the bacterial chaperone SlyD and inhibits the cell wall precursor forming enzyme MraY. Bacterial cell wall biosynthesis is an important target for antibiotics; therefore, knowledge of molecular details in the ΦX174-E lysis pathway could help to identify new mechanisms and sites of action. In this study, cell-free expression and nanoparticle technology were combined to avoid toxic effects upon ΦX174-E synthesis, resulting in the efficient production of a functional full-length toxin and engineered derivatives. Pre-assembled nanodiscs were used to study ΦX174-E function in defined lipid environments and to analyze its membrane insertion mechanisms. The conformation of the soluble domain of ΦX174-E was identified as a central trigger for membrane insertion, as well as for the oligomeric assembly of the toxin. Stable complex formation of the soluble domain with SlyD is essential to keep nascent ΦX174-E in a conformation competent for membrane insertion. Once inserted into the membrane, ΦX174-E assembles into high-order complexes via its transmembrane domain and oligomerization depends on the presence of an essential proline residue at position 21. The data presented here support a model where an initial contact of the nascent ΦX174-E transmembrane domain with the peptidyl-prolyl isomerase domain of SlyD is essential to allow a subsequent stable interaction of SlyD with the ΦX174-E soluble domain for the generation of a membrane insertion competent toxin.

Morphing of Amphipathic Helices to Explore the Activity and Selectivity of Membranolytic Antimicrobial Peptides.

Naturally occurring membranolytic antimicrobial peptides (AMPs) are rarely cell-type selective and highly potent at the same time. Template-based peptide design can be used to generate AMPs with improved properties de novo. Following this approach, 18 linear peptides were obtained by computationally morphing the natural AMP Aurein 2.2d2 GLFDIVKKVVGALG into the synthetic model AMP KLLKLLKKLLKLLK. Eleven of the 18 chimeric designs inhibited the growth of Staphylococcus aureus, and six peptides were tested and found to be active against one resistant pathogenic strain or more. One of the peptides was broadly active against bacterial and fungal pathogens without exhibiting toxicity to certain human cell lines. Solution nuclear magnetic resonance and molecular dynamics simulation suggested an oblique-oriented membrane insertion mechanism of this helical de novo peptide. Temperature-resolved circular dichroism spectroscopy pointed to conformational flexibility as an essential feature of cell-type selective AMPs.

Defining the Substrate Spectrum of the TIM22 Complex Identifies Pyruvate Carrier Subunits as Unconventional Cargos.

SummaryIn mitochondria, the carrier translocase (TIM22 complex) facilitates membrane insertion of multi-spanning proteins with internal targeting signals into the inner membrane [1, 2, 3]. Tom70, a subunit of TOM complex, represents the major receptor for these precursors [2, 4, 5, 6]. After transport across the outer membrane, the hydrophobic carriers engage with the small TIM protein complex composed of Tim9 and Tim10 for transport across the intermembrane space (IMS) toward the TIM22 complex [7, 8, 9, 10, 11, 12]. Tim22 represents the pore-forming core unit of the complex [13, 14]. Only a small subset of TIM22 cargo molecules, containing four or six transmembrane spans, have been experimentally defined. Here, we used a tim22 temperature-conditional mutant to define the TIM22 substrate spectrum. Along with carrier-like cargo proteins, we identified subunits of the mitochondrial pyruvate carrier (MPC) as unconventional TIM22 cargos. MPC proteins represent substrates with atypical topology for this transport pathway. In agreement with this, a patient affected in TIM22 function displays reduced MPC levels. Our findings broaden the repertoire of carrier pathway substrates and challenge current concepts of TIM22-mediated transport processes.

Divalent Cations and Lipid Composition Modulate Membrane Insertion and Cancer-Targeting Action of pHLIP

The pH-Low Insertion Peptide (pHLIP) has emerged as an important tool for targeting cancer cells; it has been assumed that its targeting mechanism depends solely on the mild acidic environment surrounding tumors. Here, we examine the role of Ca2+ and Mg2+ on pHLIP's insertion, cellular targeting, and drug delivery. We demonstrate that physiologically relevant concentrations of either cation can shift the protonation-dependent transition by up to several pH units toward basic pH and induce substantial protonation-independent transmembrane insertion of pHLIP at pH as high as 10. Consistent with these results, the ability of pHLIP to deliver the cytotoxic compound monomethyl-auristatin-F to HeLa cells is increased several fold in presence of Ca2+. Complementary measurements with model membranes confirmed this Ca2+/Mg2+-dependent membrane-insertion mechanism. The magnitude of this alternative Ca2+/Mg2+-dependent effect is also modulated by lipid composition—specifically by the presence of phosphatidylserine—providing new clues to pHLIP's unique tumor-targeting ability in vivo. These results exemplify the complex coupling between protonation of anionic residues and lipid-selective targeting by divalent cations, which is relevant to the general signaling on membrane interfaces.

Cholesterol promotes Cytolysin A activity by stabilizing the intermediates during pore formation

Pore-forming toxins (PFTs) form nanoscale pores across target membranes causing cell death. Cytolysin A (ClyA) from Escherichia coli is a prototypical α-helical toxin that contributes to cytolytic phenotype of several pathogenic strains. It is produced as a monomer and, upon membrane exposure, undergoes conformational changes and finally oligomerizes to form a dodecameric pore, thereby causing ion imbalance and finally cell death. However, our current understanding of this assembly process is limited to studies in detergents, which do not capture the physicochemical properties of biological membranes. Here, using single-molecule imaging and molecular dynamics simulations, we study the ClyA assembly pathway on phospholipid bilayers. We report that cholesterol stimulates pore formation, not by enhancing initial ClyA binding to the membrane but by selectively stabilizing a protomer-like conformation. This was mediated by specific interactions by cholesterol-interacting residues in the N-terminal helix. Additionally, cholesterol stabilized the oligomeric structure using bridging interactions in the protomer-protomer interfaces, thereby resulting in enhanced ClyA oligomerization. This dual stabilization of distinct intermediates by cholesterol suggests a possible molecular mechanism by which ClyA achieves selective membrane rupture of eukaryotic cell membranes. Topological similarity to eukaryotic membrane proteins suggests evolution of a bacterial α-toxin to adopt eukaryotic motifs for its activation. Broad mechanistic correspondence between pore-forming toxins hints at a wider prevalence of similar protein membrane insertion mechanisms.

The Pseudomonas aeruginosa type III secretion translocator PopB assists the insertion of the PopD translocator into host cell membranes

Many Gram-negative bacterial pathogens use a type III secretion system to infect eukaryotic cells. The injection of bacterial toxins or protein effectors via this system is accomplished through a plasma membrane channel formed by two bacterial proteins, termed translocators, whose assembly and membrane-insertion mechanisms are currently unclear. Here, using purified proteins we demonstrate that the translocators PopB and PopD in Pseudomonas aeruginosa assemble heterodimers in membranes, leading to stably inserted hetero-complexes. Using site-directed fluorescence labeling with an environment-sensitive probe, we found that hydrophobic segments in PopD anchor the translocator to the membrane, but without adopting a typical transmembrane orientation. A fluorescence dual-quenching assay revealed that the presence of PopB changes the conformation adopted by PopD segments in membranes. Furthermore, analysis of PopD's interaction with human cell membranes revealed that PopD adopts a distinctive conformation when PopB is present. An N-terminal region of PopD is only exposed to the host cytosol when PopB is present. We conclude that PopB assists with the proper insertion of PopD in cell membranes, required for the formation of a functional translocon and host infection.

Structure-Activity Correlations of Amphipathic Cationic AMPs: MD Simulations and Antibacterial Measurement

Bacterial drug resistance is one of the largest issues that impede the development of new antibiotics. Since their discovery in multiple hosts, antimicrobial peptides (AMPs) appear to be a viable alternative to antibiotics. Here, we report structure-activity correlations of an amphipathic cationic peptide P1, KKLIKKILKIL, and its analogs, that target bacterial membrane unlike the antibiotics that target intracellular machineries. We performed molecular dynamics simulations on P1 and its analogs to study their insertion into a model lipid bilayer. We also measured the antibacterial activities of P1 and its analogs on a wildtype E. coli and a resistant strain evolved against P1. Combining experiment and molecular dynamics simulations, we show that: (i) neutralization of the C-terminal L of P1 enhances both membrane insertion and antibacterial activity; (ii) reduction of charge and hydrophobicity in P2, KKLAKEILKIL (with underlined amino acid substitutions) drastically reduces the antibacterial activity; (iii) P3 with C-terminal charge neutralization remains susceptible to resistance mechanism evolved against P1; (iv) however, increase in electrostatic interaction with the lipid in P4, RRLIRRILRIL with K to R substitutions, renders protection against bacterial resistance. In summary, combination of molecular dynamics simulations and antibacterial activity measurement provide insight into the mechanism of membrane insertion of amphipathic cationic AMPs and offer guidelines to increase their activity and decrease their susceptibility to bacterial resistance.

Membrane protein insertion through a mitochondrial β-barrel gate.

The biogenesis of mitochondria, chloroplasts, and Gram-negative bacteria requires the insertion of β-barrel proteins into the outer membranes. Homologous Omp85 proteins are essential for membrane insertion of β-barrel precursors. It is unknown if precursors are threaded through the Omp85-channel interior and exit laterally or if they are translocated into the membrane at the Omp85-lipid interface. We have mapped the interaction of a precursor in transit with the mitochondrial Omp85-channel Sam50 in the native membrane environment. The precursor is translocated into the channel interior, interacts with an internal loop, and inserts into the lateral gate by β-signal exchange. Transport through the Omp85-channel interior followed by release through the lateral gate into the lipid phase may represent a basic mechanism for membrane insertion of β-barrel proteins.

Lysenin Toxin Membrane Insertion Is pH-Dependent but Independent of Neighboring Lysenins

Pore-forming toxins form a family of proteins that act as virulence factors of pathogenic bacteria, but similar proteins are found in all kingdoms of life, including the vertebrate immune system. They are secreted as soluble monomers that oligomerize on target membranes in the so-called prepore state; after activation, they insert into the membrane and adopt the pore state. Lysenin is a pore-forming toxin from the earthworm Eisenida foetida, of which both the soluble and membrane-inserted structures are solved. However, the activation and membrane-insertion mechanisms have remained elusive. Here, we used high-speed atomic force microscopy to directly visualize the membrane-insertion mechanism. Changing the environmental pH from pH 7.5 to below pH 6.0 favored membrane insertion. We detected a short α-helix in the soluble structure that comprised three glutamic acids (Glu92, Glu94, and Glu97) that we hypothesized may represent a pH-sensor (as in similar toxins, e.g., Listeriolysin). Mutant lysenin still can form pores, but mutating these glutamic acids to glutamines rendered the toxin pH-insensitive. On the other hand, toxins in the pore state did not favor insertion of neighboring prepores; indeed, pore insertion breaks the hexagonal ordered domains of prepores and separates from neighboring molecules in the membrane. pH-dependent activation of toxins may represent a common feature of pore-forming toxins. High-speed atomic force microscopy with single-molecule resolution at high temporal resolution and the possibility of exchanging buffers during the experiments presents itself as a unique tool for the study of toxin-state conversion.

Insights into Cotranslational Membrane Protein Insertion by Combined LILBID-Mass Spectrometry and NMR Spectroscopy.

Cotranslational insertion of membrane proteins into defined nanoparticle membranes has been developed as an efficient process to produce highly soluble samples in native-like environments and to study lipid-dependent effects on protein structure and function. Numerous examples of the structural and functional characterization of transporters, ion channels, or G-protein-coupled receptors in cotranslationally formed nanodisc complexes demonstrate the versatility of this approach, although the basic underlying mechanisms of membrane insertion are mainly unknown. We have revealed the first aspects of the insertion of proteins into nanodiscs by combining cell-free expression, noncovalent mass spectrometry, and NMR spectroscopy. We provide evidence of cooperative insertion of homo-oligomeric complexes and demonstrate the possibility to modulate their stoichiometry by modifying reaction conditions. Additionally, we show that significant amounts of lipid are released from the nanodiscs upon insertion of larger protein complexes.

Mechanisms of Tail-Anchored Membrane Protein Targeting and Insertion.

Proper localization of membrane proteins is essential for the function of biological membranes and for the establishment of organelle identity within a cell. Molecular machineries that mediate membrane protein biogenesis need to not only achieve a high degree of efficiency and accuracy, but also prevent off-pathway aggregation events that can be detrimental to cells. The posttranslational targeting of tail-anchored proteins (TAs) provides tractable model systems to probe these fundamental issues. Recent advances in understanding TA-targeting pathways reveal sophisticated molecular machineries that drive and regulate these processes. These findings also suggest how an interconnected network of targeting factors, cochaperones, and quality control machineries together ensures robust membrane protein biogenesis.

Simulation of lipid-protein interactions with the CgProt force field

The effect of lipid-protein interactions on membrane proteins and their function is emerging as an important area in biophysics. The recently developed CgProt force field is used to explore molecular level interactions in peptides and proteins through coarse-grained molecular dynamics simulations in the presence of the lipid bilayer environment. The mechanism of membrane insertion was examined for designed helical peptides WALP27: GWW(LA)₁₀LWWA and LS3: (LSSLLSL)₃. WALP27 adopts a transmembrane conformation while LS3 adopts interfacial and transmembrane orientations in independent simulations from different starting conditions. A total of 13 peptide insertion events were observed in unbiased molecular dynamics simulations of LS3 and WALP. Each case proceeded via the charged N-terminus crossing the bilayer. In the equilibrated conformations, the C-terminus resides in the head group region of the DOPC bilayer, while the N-terminus submerges deep into the carbonyl region. These findings are consistent with recent evidence of the attractive nature of positive charges for the membrane interface. The role of lipid interactions in the native environment of a membrane protein was explored by simulation of a 12-transmembrane helix multidrug transporter, P-glycoprotein. Analysis of the lipid density in a POPC: POPE bilayer containing 20% cholesterol revealed an annular solvation shell of phosphatidyl choline and cholesterol that diffuses with the protein in the membrane. The inward-facing state of P-glycoprotein undergoes a conformational transition to the closed state, corroborating a structural model for the ATP-bound state recently generated by homology modeling and molecular dynamics simulation.