112 research outputs found

    Structure-Based High-Throughput Epitope Analysis of Hexon Proteins in B and C Species Human Adenoviruses (HAdVs)

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    Human adenoviruses (HAdVs) are the etiologic agent of many human infectious diseases. The existence of at least 54 different serotypes of HAdVs has resulted in difficulties in clinical diagnosis. Acute respiratory tract disease (ARD) caused by some serotypes from B and C species is particularly serious. Hexon, the main coat protein of HAdV, contains the major serotype-specific B cell epitopes; however, few studies have addressed epitope mapping in most HAdV serotypes. In this study, we utilized a novel and rapid method for the modeling of homologous proteins based on the phylogenetic tree of protein families and built three-dimensional (3D) models of hexon proteins in B and C species HAdVs. Based on refined hexon structures, we used reverse evolutionary trace (RET) bioinformatics analysis combined with a specially designed hexon epitope screening algorithm to achieve high-throughput epitope mapping of all 13 hexon proteins in B and C species HAdVs. This study has demonstrated that all of the epitopes from the 13 hexon proteins are located in the proteins' tower regions; however, the exact number, location, and size of the epitopes differ among the HAdV serotypes

    A THEORETICAL INVESTIGATION EXAMINING DNA CONFORMATIONAL CHANGES AND THEIR EFFECTS ON GLYCOSYLASE FUNCTION

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    Glycosylase enzymes initiate the process of base excision repair (BER) in order to prevent the irreversible modification of the genome. In the BER process a damaged DNA base is recognized, removed from the DNA sequence, and then the remaining abasic site is repaired. Glycosylase enzymes are responsible for the base recognition mechanism and catalysis of the base excision. One of the most studied glycosylase superfamilies is uracil DNA glycosylase (UDG). The UDG superfamily has demonstrated specificity for excising uracil, which is the deamination product of cytosine, from DNA sequences of prokaryotes and eukaryotes. Mismatch-specific uracil DNA glycosylase (MUG) is a member of the UDG superfamily, and interestingly has shown specificity for both uracil and xanthine bases. The following dissertation provides an anlaysis on the recognition mechanism of E. coli MUG for deaminated DNA bases. Glycosylase enzymes require the damaged base to be flipped out of the base stack, and into an active site for catalysis of the N-glycosidic cleavage. Typically, recognition of substrates by enzymes is characterized by binding affinities, but in the following work the binding of E.Coli MUG is broken down into contributions from the base flipping and enzyme binding equilibria. Since DNA conformational changes play a large role in UDG systems, the robustness of molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) free energy method was evaluated for a DNA conformational change. The A-form to B-form DNA conformational free energy differences were calculated using MM/PBSA, and compared with free energy differences determined with a more rigorous umbrella sampling method. MM/PBSA calculations of the free energy difference between A-form and B-form DNA are shown to be in very close agreement with the PMF result determined using an umbrella sampling approach. The sensitivity to solvent model and force field used during conformational sampling was also established for the MM/PBSA free energies. In order to determine the influence of base flipping conformational changes on the MUG recognition process, PMF profiles were generated for each of the damaged bases (uracil, xanthine, oxanine, inosine). Agreement was displayed between the base pair stability trends from the umbrella sampling, and the enzyme activities from experiment. Interaction energies and structural analyses were used to examine the MUG enzyme, which revealed regions of the active site critical for binding xanthine and uracil substrates. Site-directed mutagenesis experiments were performed on MUG to determine the role of specific amino acids in the recognition mechanism. Mutations were studied further through modeling and molecular dynamics (MD) simulations of the unbound and bound proteins

    Molecular dynamics simulations of adhesion and nanoidentation of gallium arsenide

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    The purpose of this dissertation is to investigate the nanoscale hardness of gallium arsenide thin films and the elastic-plastic behavior of gallium arsenide under an indenter. These investigations were carried out using molecular dynamics (MD) simulations. The simulations are based on interatomic potentials that accurately reproduce many properties of bulk GaAs. The MD simulations performed required scalable and efficient algorithms for implementation on large parallel computers. Nanoindentation simulations were performed using an ideal indenter that was held rigid during the simulation. To reduce the transient effects due to loading, the traversal of the indenter was interrupted periodically to allow the substrate to relax. Load-displacement curves were calculated and Vickers hardness and Young’s modulus were computed from the curves. The damage caused by the indenter was characterized in three ways. The material deposited on the surface was compared to bulk amorphous GaAs and found to be structually similar, indicating that the material underwent solid-state amorphization under the indenter. Analysis of energetic atoms beneath the surface suggested the presence of dislocation loops. A centrosymmetry method was applied to characterize these defects. It was found that the method used did not perform adequately in the presence of amorphized material. Pressure distributions were calculated and atomic configurations were plotted to determine if subsurface microcracking due to the indentation was present. No indication of microcracking or pore formation was found. Adhesion between the tip and substrate was also studied. The effect of the tip-surface attraction was studied for a modified Vickers indenter with a small flat surface instead of an atomically sharp tip. For indentations less than the yield point in GaAs, the bond formation between the tip and the surface led to nonelastic deformation of the surface layer, while the layers undeneath the surface behaved in a purely elastic fashion. Through a series of small indenter traversals, the yield point of GaAs was determined to be 0.6 µN

    Molecular Dynamics Simulation

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    Condensed matter systems, ranging from simple fluids and solids to complex multicomponent materials and even biological matter, are governed by well understood laws of physics, within the formal theoretical framework of quantum theory and statistical mechanics. On the relevant scales of length and time, the appropriate ‘first-principles’ description needs only the Schroedinger equation together with Gibbs averaging over the relevant statistical ensemble. However, this program cannot be carried out straightforwardly—dealing with electron correlations is still a challenge for the methods of quantum chemistry. Similarly, standard statistical mechanics makes precise explicit statements only on the properties of systems for which the many-body problem can be effectively reduced to one of independent particles or quasi-particles. [...

    Molecular simulation studies of the prion protein: from disease-linked variants to ligand binding

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    Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neu-rodegenerative disorders (198). The crucial event in the development of these diseases is the conformational change of a membrane bound protein, the cellular PrPC in Figure 3.1, into a disease associated, bril-forming isoform (199). Despite their rare incidence, TSEs have captured very large attention from the scienti c community due to the unorthodox mechanism by which prion diseases are transmitted..

    Theoretical and Experimental Investigations of the Interaction of Proteins and Nanoparticles with Biological Membranes

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    Biomolecular processes related to the interaction of proteins, AuNPs and biological membranes are studied in this thesis. In particular, computational methods were developed, implemented and validated. To characterize the influence of antimicrobial peptides and AuNPs on a membrane, black lipid bilayer experiments were performed. To understand interactions of certain AuNPs with the hERG ion channel, complex formation between these two was studied using atomistic simulations

    Computational Approaches to Simulation and Analysis of Large Conformational Transitions in Proteins

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    abstract: In a typical living cell, millions to billions of proteins—nanomachines that fluctuate and cycle among many conformational states—convert available free energy into mechanochemical work. A fundamental goal of biophysics is to ascertain how 3D protein structures encode specific functions, such as catalyzing chemical reactions or transporting nutrients into a cell. Protein dynamics span femtosecond timescales (i.e., covalent bond oscillations) to large conformational transition timescales in, and beyond, the millisecond regime (e.g., glucose transport across a phospholipid bilayer). Actual transition events are fast but rare, occurring orders of magnitude faster than typical metastable equilibrium waiting times. Equilibrium molecular dynamics (EqMD) can capture atomistic detail and solute-solvent interactions, but even microseconds of sampling attainable nowadays still falls orders of magnitude short of transition timescales, especially for large systems, rendering observations of such "rare events" difficult or effectively impossible. Advanced path-sampling methods exploit reduced physical models or biasing to produce plausible transitions while balancing accuracy and efficiency, but quantifying their accuracy relative to other numerical and experimental data has been challenging. Indeed, new horizons in elucidating protein function necessitate that present methodologies be revised to more seamlessly and quantitatively integrate a spectrum of methods, both numerical and experimental. In this dissertation, experimental and computational methods are put into perspective using the enzyme adenylate kinase (AdK) as an illustrative example. We introduce Path Similarity Analysis (PSA)—an integrative computational framework developed to quantify transition path similarity. PSA not only reliably distinguished AdK transitions by the originating method, but also traced pathway differences between two methods back to charge-charge interactions (neglected by the stereochemical model, but not the all-atom force field) in several conserved salt bridges. Cryo-electron microscopy maps of the transporter Bor1p are directly incorporated into EqMD simulations using MD flexible fitting to produce viable structural models and infer a plausible transport mechanism. Conforming to the theme of integration, a short compendium of an exploratory project—developing a hybrid atomistic-continuum method—is presented, including initial results and a novel fluctuating hydrodynamics model and corresponding numerical code.Dissertation/ThesisDoctoral Dissertation Physics 201

    Advanced adaptive resolution methods for molecular simulation

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