1,720,983 research outputs found
Molecular Dynamics Simulations of Ion Permeation in Human Voltage-Gated Sodium Channels
Get PDFThe recent determination of cryo-EM structures of voltage-gated sodium (Nav) channels has revealed many details of these proteins. However, knowledge of ionic permeation through the Nav pore remains limited. In this work, we performed atomistic molecular dynamics (MD) simulations to study the structural features of various neuronal Nav channels based on homology modeling of the cryo-EM structure of the human Nav1.4 channel and, in addition, on the recently resolved configuration for Nav1.2. In particular, single Na+ permeation events during standard MD runs suggest that the ion resides in the inner part of the Nav selectivity filter (SF). On-the-fly free energy parametrization (OTFP) temperature-accelerated molecular dynamics (TAMD) was also used to calculate two-dimensional free energy surfaces (FESs) related to single/double Na+ translocation through the SF of the homology-based Nav1.2 model and the cryo-EM Nav1.2 structure, with different realizations of the DEKA filter domain. These additional simulations revealed distinct mechanisms for single and double Na+ permeation through the wild-type SF, which has a charged lysine in the DEKA ring. Moreover, the configurations of the ions in the SF corresponding to the metastable states of the FESs are specific for each SF motif. Overall, the description of these mechanisms gives us new insights into ion conduction in human Nav cryo-EM-based and cryo-EM configurations that could advance understanding of these systems and how they differ from potassium and bacterial Nav channels
Polymorphism at 129 dictates metastable conformations of the human prion protein N-terminal β-sheet
Get PDFWe study the thermodynamic stability of the native state of the human prion protein using a new free-energy method, replica-exchange on-the-fly parameterization. This method is designed to overcome hidden-variable sampling limitations to yield nearly error-free free-energy profiles along a conformational coordinate. We confirm that all four (M129V, D178N) polymorphs have a ground-state conformation with three intact β-sheet hydrogen bonds. Additionally, they are observed to have distinct metastabilities determined by the side-chain at position 129. We rationalize these findings with reference to the prion “strain” hypothesis, which links the variety of transmissible spongiform encephalopathy phenotypes to conformationally distinct infectious prion forms and classifies distinct phenotypes of sporadic Creutzfeldt-Jakob disease based solely on the 129 polymorphism. Because such metastable structures are not easily observed in structural experiments, our approach could potentially provide new insights into the conformational origins of prion diseases and other pathologies arising from protein misfolding and aggregation.Fil: Paz, Sergio Alexis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Drexel University; Estados UnidosFil: Vanden-Eijnden, Eric. University of New York; Estados UnidosFil: Abrams, Cameron F.. Drexel University; Estados Unido
Molecular simulations of chaperonins
Get PDFChaperonins are a class of cage-like molecular machines that assist the folding of polypeptides by binding and releasing non-native substrates into their inner cavity, where sequestered from the medium the substrate can fold to its native state. The main goal of this work is to understand the structure-function relationship of chaperonin. Here we study two major aspects of this relationship: (1) thermodynamics of protein folding inside the chaperonin cavity and (2) conformational changes of the E. coli chaperonin GroEL in its reaction cycle. We have studied the thermodynamics of protein folding, confined in the chaperonin cavity using the simple HP model of protein undergoing the coil-to-globule transition. Using the Wang-Landau method we have quantified the increase in thermal stability of a native state in a spherical confining geometry, measured as an increase in melting temperature with decreasing radius of confining sphere. We are the first to show that the t -> r transition in GroEL subunit occurs spontaneously using unbiased molecular dynamics simulation. The transition pathway is shown to lie along low frequency quasi-harmonic modes of vibration. We are also the first to observe the spontaneous insertion of Ala480 into the empty nucleotide binding pocket, required for negative interring cooperativity.Ph.D., Chemical Engineering -- Drexel University, 200
Molecular simulations of monomeric sarcosine oxidase
Get PDFMonomeric sarcosine oxidase (MSOX) is a flavoenzyme that catalyzes the oxidation of sarcosine to form formaldehyde, glycine and hydrogen peroxide. MSOX also reduces molecular oxygen. There is no channel detected by experiment that transports oxygen from the protein surface to the buried active site for oxygen reduction. The main purpose of this work is to determine the oxygen transport channels in MSOX using molecular simulations. Here we used two methods: Molecular Dynamics (MD) and Single-sweep free energy reconstruction. Our results show that there exist two different pathways to the oxygen activation site: the so called si-side and re-side entry pathways. Between these two, the si-side entry pathway is more thermodynamically favorable. This highlights the efficiency of MSOX because the substrate and O2 access the catalytic center via two distinct pathways.M.S., Chemical and Biological Engineering -- Drexel University, 201
Kinetics of Oxygen Transport in Monomeric Sarcosine Oxidase
Get PDFFlavin-containing oxidases are a class of proteins which use oxygen to regenerate the oxidized state of the isoalloxazine ring in flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) after it has been reduced by substrate oxidation. Though well characterized experimentally, many questions linger regarding how small molecules access the active site as well as where oxygen activation occurs in flavin-containing oxidases. A prototypical member of this family is monomeric sarcosine oxidase (MSOX), and it is perhaps the most well studied. Despite knowing which features are essential for catalysis as well as the location of the substrate binding site, it is unclear how oxygen accesses the site since the only clear entryway can be partially blocked by the larger substrate sarcosine. As such, two competing mechanisms have gained attention centering around the order by which ligands enter the binding site. In this thesis, we detail the use of all atom molecular dynamics (MD) studies to identify how oxygen accesses the MSOX active site, as well as characterize the resulting kinetic network. We use the single sweep method to identify four potential routes for oxygen to travel from the surface of MSOX to the active site. Then, using Markovian milestoning with Voronoi tessellations (MMVT), we refine the pathways identified in single sweep and develop a Markov state model describing the kinetics of oxygen entry and exit. We calculate entry and exit mean first passage times (MFPT) for oxygen from this model, which are used to compute second order rate constants for entry and first order rate constants for exit. Our calculated rate constants and mechanisms show that the presence of a substrate-mimicking inhibitor markedly influences the kinetics of oxygen entry and exit. The bound competitive inhibitor changes the protein structure sufficiently to shut down almost all major oxygen channels save one, which it opens, speeding entry but greatly slowing down oxygen exit, relative to the substrate-free enzyme. This means that our kinetic analysis predicts oxygen exhibits a longer residence time within MSOX when a substrate-like ligand is present. This supports the so-called "modified ping-pong" mechanism, in agreement with previous experimental results, thus lending validity to our approach. Furthermore, our computed second-order entry rate constants are larger by about an order of magnitude than are experimentally determined oxygen consumption rate constants. Since oxygen consumption combines the processes of entry and electron transfer, we conclude that of these two, entry is not rate-limiting in the overall catalytic cycle, regardless of whether or not a substrate-like ligand is bound. Finally, because this work represents the first test of the MMVT approach for comparing kinetics of ligand entry for an enzyme in two distinct states, we not surprisingly uncovered inefficiencies in the approach. We tested one idea for gaining efficiency based on the "finite-temperature" string method, in which transport channels can be determined and kinetically characterized on-the-fly, rather than sequentially. Our results indicate that more research in that area is needed.Ph.D., Chemical Engineering -- Drexel University, 201
Molecular simulations and modeling of HIV-1 gp41 membrane spanning domain (MSD) in a model viral bilayer
Get PDFHIV-1 envelope protein complexes known as “spikes” are trimers of gp120 and gp41 that mediate fusion and infection to target cell membranes. The membrane spanning domain (MSD) of gp41 contains several highly conserved residues important for fusion, however the structure and function of MSD are only partly understood. All-atom simulations can elucidate how the conserved residues affect MSD structure to provide atomistic insight into HIV-1 fusion.
Extensive molecular dynamics (MD) of monomeric HIV-1 gp41 MSD in model viral bilayers was used to investigate the conserved midspan arginine and the requirement of cholesterol for fusion. All wild-type peptides were α-helical, remained membrane-spanning, and solvated their midspan arginines with a water defect that was independent of cholesterol. However, the simulations indicate that cholesterol may allow the spike to localize the water defect and to control the tilt of the helices.
The dynamics of the model viral bilayer with ∼50% cholesterol was explored with 3 systems and simulated for up to 10 μs to explore the phase space of configurations, an order of magnitude greater than previous studies. This timescale allowed observation of diffusive motion and calculated diffusion coefficients agreed with experiments.
Oligomeric forms of the MSD were then created to examine the GXXXG motif, known for helical, transmembrane, dimer interactions, but conserved in the MSD of the trimeric gp41. The lowest-energy trimeric MSD with interacting GXXXG residues could not mediate trimerization in a bilayer on 100 ns timescales. However, the lowest energy MSD dimer remained associated on similar timescales, suggesting a dimer form of the MSD during fusion.
Finally, the trimeric MSD was stabilized by addition of the trimeric crystal structure of the gp41 membrane proximal external region (MPER). Simulation of the MPER-MSD trimer for 11 μs showed relaxation towards a different, stable configuration in which the GXXXG motifs were not interacting but the cholesterol recognition motif (CRAC) sequestered water and cholesterol. This is the first simulation of a model of trimeric MPER-MSD in a cholesterol-containing bilayer and it may represent a point between the prefusion and the prefusion intermediate experimental trimeric structures.Ph.D., Chemical Engineering -- Drexel University, 201
Novel simulation methods for characterizing conformational changes in nicotinic acetylcholine receptor analogues and beta-2-microglobulin
No full textMolecular simulation tools and protocols were developed for characterizing conformational changes in several proteins: beta2-microglobulin (beta2M), the nicotinic acetylcholine receptor (nAChR), the acetylcholine binding protein (AChBP), and an alpha7-nicotinic acetylcholine receptor/acetylcholine binding protein chimera (alpha7-nAChR-AChBP chimera).The normal to amyloidogenic transformation of beta2M was analyzed using our implementation of on-the-y string method (OFSM) in collective variables (CVs), an algorithm for mapping free energy surfaces along a reaction coordinate. We show that the protonation state of two histidine residues is of key importance in mediating this transition. Also, our predicted free energy barriers for this transition agree with similar experimental evidence.Next, we constructed a membrane-embedded model of a complete nAChR based on the structure determined by Unwin via electron image reconstruction [Unwin, J Mol Biol 346:967, 2005]. Temperature accelerated molecular dynamics (TAMD), a method for rapid sampling of CV space, was used to examine the e ects of CV selection and degree of TAMD acceleration on conformations visited during simulation. These results showed that TAMD explores conformational space roughly an order-of-magnitude faster than traditional molecular dynamics (MD) simulation, with correct choices for CVs and other parameters. Unfortunately, these experiments also revealed that the 4 A resolution of this structure is insufficient for application of extensive accelerated sampling techniques.The AChBP is often used as a surrogate for the nAChR because of its similarity to the nAChR extracellular domain (ECD) and the availability of high-resolution structures. A challenge in conformational analysis of AChBP structures is that apo and ligand-bound conformations for the multitude of available structures are nearly superimposable. To address this, we developed a new technique that uses an alignment-free statistical algorithm to assign state determining CVs. This method is successful at discriminating partial from full agonists via analysis of AChBP crystal structures and is a generally applicable method for efficient selection of CVs for use in molecular simulation.Finally we examine two recent crystal structures of an alpha7-nAChR-AChBP chimera in apo and agonist-bound conformations [Li, Nat. Neurosci. 1253:9, 2011]. We developed a new protocol using targeted molecular dynamics (TrMD) followed by restrained MD equilibration to synthesize the conformation of the ligand encounter complex (the conformation generated just after ligand binding occurs) from these structures. We show that this complex is stable on MD time scales and exhibits features of both apo and agonist-bound conformations. Ligand binding energies are computed for the ligand encounter complex and agonist-bound states that are similar to experimental values for the alpha7-nicotinic acetylcholine receptor (alpha7-nAChR). Finally, we discuss preliminary results of an OFSM simulation for the apo and ligand-bound alpha7-nAChR-AChBP chimera.Ph.D., Chemical Engineering -- Drexel University, 201
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Molecular Simulations of 12-Hydroxystearic Acid and Its Derivatives in Organic Solvent
No full text12-Hydroxystearic acid (12HSA) and its derivatives are well-known organogelators, and they play critical roles in a variety of applications. Under gelating conditions, 12HSA forms a three-dimensional network of fibers with lateral fibril dimensions on the submicron scale. It is hypothesized that layers of 12HSA molecules stack to produce a multilamellar structure with alignment of 12-hydroxyl groups that form a chain of hydrogen bonds, and the twist of the fibers is due to the nonlinearity in the carbon chain at the 12th carbon. Furthermore, the sense of twist is sensitive to both the size of the carboxylate counterion and the chirality at the 12th carbon. However, the details underlying these differences are difficult to infer from low-resolution information available from, for example, X-ray scattering. In this thesis, we detail the use of molecular dynamics (MD) simulations to identify key functional group interactions that impact the morphology of aggregates of 12HSA and its derivatives, and to assess the importance of optical purity at the 12th carbon and form of the head-group on aggregate structure. First, in order to assess the aggregate morphology, we conducted MD simulations on microsecond-long time scales for (R)-12HSA at 12.5 wt % in explicit hexane solvent. Self-assembly was accelerated by using a modified force field to prohibit alkane chain dihedral gauche states and then verified by continuation using standard force-field parameters. In three independent simulations, acceleration using a ``gauche-less'' force field resulted in self-assembled ordered aggregates through formation of polarized five- and six-membered rings between inter-12-hydroxyl groups and head-to-head carboxylic acid dimerization. When subjected to the unmodified dihedral force field, two of the three structures remained stable after 1 microsecond of MD. Stable structures exhibited a ``ring-of-rings'' motif, composed of two six-membered acetic acid-dimerized ring bundles with six satellite rings, while unstable structures did not. These structures displayed scattering peaks that agreed with experiment, which suggests this ``ring-of-rings'' structure could be a primary feature of (R)-12HSA in organic solvents. Next, we studied the effect of optical purity on the aggregate structures of 12HSA. We conducted microsecond long MD simulations on (1) (R)-12HSA, (2) (S)-12HSA, and (3) a 50/50 racemic mixture, each solvated at 12.5 wt % in explicit hexane. The ``full-cycling'' method described previously was used for these simulations. Similarly to the optically pure (R)-12HSA aggregates, (R)-12HSA aggregates observed the same ring-of-rings motif. However, the chirality at the 12th carbon dictates the overall twist of the rings and thereby the handedness of the rings. Racemic mixtures did not produce stable ordered aggregates, likely due to insufficient enantiomerically pure ring formation. Next, we analyze the impact of the head-group counterion on aggregate morphology by testing systems of optically pure 12-hydroxystearate with both lithium-carboxylate and sodium-carboxylate head-groups. Due to difficulties with the TraPPE-UA force-field in observing correct ion-carboxylate dimer complexes predicted by quantum mechanical results that showed the high stability of dimer complexes compared to undimerized states, we created predefined dimer complexes based on structures from quantum mechanical calculations. The lithium-carboxylate system exhibited two stable dimer forms: (1) centrally symmetric and (2) diagonally symmetric, while the sodium-carboxylate system only had a diagonally symmetric dimer. We found that similar to the acid systems, all of the ion 12-hydroxystearate systems had inter-hydroxyl hydrogen bonds that were oriented in a ringlike fashion. However, we found that the centrally symmetric dimer allowed for a higher frequency of inter-hydroxyl hydrogen bonds than that of the diagonally symmetric systems, due to its ability for the head groups to pack more easily. Finally, we attempted to find the self-assembly mechanism of 12HSA and its derivatives. We opted to use umbrella sampling with a 1D collective variable (CV) that would influence the alkane dihedral configuration. Multiple CVs were studies, but the most practical CV developed was an indirect control of the alkane dihedral conformation by controlling the distance between carbons 1 and 12. This method had difficulty predicting a free-energy minimum due to time scale constraints. Further enhanced sampling methods should be tried, such as replica-exchange MD (REMD).Ph.D., Chemical Engineering -- Drexel University, 201
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