Дисертації: "Proteins Molecular Dynamics Computational Biophysics"

1

Rigoli, Marta. "The structure-dynamics-function relation in proteins: bridging all-atom molecular dynamics, experiments, and simplified models." Doctoral thesis, Università degli studi di Trento, 2022. https://hdl.handle.net/11572/330870.

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Proteins are one of the most studied biological molecules of the last decades. A great amount of experimental techniques provide to researchers direct or indirect informations on proteins structure and function. In silico simulations can be used as a “computational microscope” giving the possibility to observe protein dynamic properties at atomistic resolution. In this work, various applications of computational methods to biological systems are presented. In particular, all-atom Molecular Dynamics (MD) simulations were employed to investigate the behaviour of proteins at atomstic resolution. The term “Molecular Dynamics” is usually referred to computational methods used for the simulation of classical many-body systems. These techniques are applied to microscopic systems and they represent a powerful approach for the study of physical processes, providing a tool for their interpretation. They have been widely used in the past decades to elucidate a large variety of molecular processes in different fields such as solid state physics, material science, chemistry, biochemistry and biophysics. Here, all-atom MD simulations were employed to observe equilibrium properties of several biologically relevant proteins. This allowed us to direct perform a comparison of molecular mechanisms occurring at the atomistic level as obtained from in silico studies with experimental data, which usually describe processes at larger length and time scales. These MD simulations were also meant as a starting point for the construction of simplified models, as they were processed through coarse-graining procedures to extrapolate crucial systems features, such as informative protein sites, on the basis of information theory approaches. Specifically we studied the dynamics of pembrolizumab, a humanized immunoglobulin of type G4 (IgG4) used as a therapeutic antibody. It is employed for the treatment of lung cancer, melanoma, stomach and head cancer and Hodgkin’s lymphoma. This antibody interacts with the programmed cell death protein 1 (PD-1) receptor, blocking the suppression of the immune response during cancer development. The studied systems are three: the apo state of pembrolizumab, the holo state (i.e. pembrolizumab bound to PD-1) and the glycosylated apo configuration. Each configuration was simulated for 2μs, for a total of 6μs. The analysis of the trajectories was carried out by combining standard structural analysis techniques and information theory-based measures of correlation. From MD trajectories we could extract valuable informations on the connectivity that exists among the structural domains that compose the antibody structure. Moreover, it was possible to infer which regions are involved in the structural rearrangement in the case of the antigen binding. We could observe that the presence of the antigen reduces the conformational variability of the molecule giving a greater stability to it. The second studied system is the P53 protein complex. In this case we focused on the tetramerization domain (TD) region that is composed by 2 identical dimers and has the function of bringing together the four monomers of the p53 complex. Starting from the observation that in case of the mutation of residue R337 several pathologies are developed in humans, we constructed computational models to reproduce the dynamics of the mutants and investigate their behaviour in silico. We performed simulations for a total of 16 μs divided in 8 different cases. In the first part of the study the wild type (WT) protein was compared to the R337C and the R337H mutant in three different protonation states: delta protonated Histidine, epsilon protonated Histidine ad double protonated Histidine. In the second part of the study we highlighted the differences between the WT configuration and three rationally designed mutants: R337D-352D, 337R-D352R, R337D-D352R. In this part of the investigation, the importance of the electrostatic interaction between residues R337 and D352 in the stability of the tetramerization do- main was discussed. Furthermore, we matched the obtained computational results of p53 tetramerization domain with functional experiments in yeasts (performed in collaboration with the CIBIO department) of all the simulated forms. The third simulated protein is the zinc sensing transcriptional repressor (CzrA), an homodimeric protein that binds DNA in Staphylococcus aureus. All-atom MD simulations of two different configurations were performed for a total of 4μs, the first one is the WT apo protein while the second is the WT holo system, where the protein is complexed with two Zn ions. In this case, in addition to standard analysis techniques, we applied the mapping entropy minimization protocol to highlight the most informative protein regions, from the perspective of information theory. Finally, our in silico results were compared to available NMR data of the protein itself.

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2

Parton, Daniel L. "Pushing the boundaries : molecular dynamics simulations of complex biological membranes." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:7ab91b51-a5ae-46b4-b6dc-3f0dd3f0b477.

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A range of simulations have been conducted to investigate the behaviour of a diverse set of complex biological membrane systems. The processes of interest have required simulations over extended time and length scales, but without sacrifice of molecular detail. For this reason, the primary technique used has been coarse-grained molecular dynamics (CG MD) simulations, in which small groups of atoms are combined into lower-resolution CG particles. The increased computational efficiency of this technique has allowed simulations with time scales of microseconds, and length scales of hundreds of nm. The membrane-permeabilizing action of the antimicrobial peptide maculatin 1.1 was investigated. This short α-helical peptide is thought to kill bacteria by permeabilizing the plasma membrane, but the exact mechanism has not been confirmed. Multiscale (CG and atomistic) simulations show that maculatin can insert into membranes to form disordered, water-permeable aggregates, while CG simulations of large numbers of peptides resulted in substantial deformation of lipid vesicles. The simulations imply that both pore-forming and lytic mechanisms are available to maculatin 1.1, and that the predominance of either depends on conditions such as peptide concentration and membrane composition. A generalized study of membrane protein aggregation was conducted via CG simulations of lipid bilayers containing multiple copies of model transmembrane proteins: either α-helical bundles or β-barrels. By varying the lipid tail length and the membrane type (planar bilayer or spherical vesicle), the simulations display protein aggregation ranging from negligible to extensive; they show how this biologically important process is modulated by hydrophobic mismatch, membrane curvature, and the structural class or orientation of the protein. The association of influenza hemagglutinin (HA) with putative lipid rafts was investigated by simulating aggregates of HA in a domain-forming membrane. The CG MD study addressed an important limitation of model membrane experiments by investigating the influence of high local protein concentration on membrane phase behaviour. The simulations showed attenuated diffusion of unsaturated lipids within HA aggregates, leading to spontaneous accumulation of raft-type lipids (saturated lipids and cholesterol). A CG model of the entire influenza viral envelope was constructed in realistic dimensions, comprising the three types of viral envelope protein (HA, neuraminidase and M2) inserted into a large lipid vesicle. The study represents one of the largest near-atomistic simulations of a biological membrane to date. It shows how the high concentration of proteins found in the viral envelope can attenuate formation of lipid domains, which may help to explain why lipid rafts do not form on large scales in vivo.

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3

Hirst-Dunton, Thomas Alexander. "Using molecular simulations to parameterize discrete models of protein movement in the membrane." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:893568e9-696f-47e7-8495-59ecfb810459.

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The work presented in this thesis centres on the development of a work-flow in which coarse-grained molecular dynamics (MD) simulations of a planar phospholipid bilayer, containing membrane proteins, is used to parameterize a larger-scale simplified bilayer model. Using this work-flow, repeat simulations and simulations of larger systems are possible, better enabling the calculation of bulk statistics for the system. The larger-scale simulations can be run on commercial hardware, once the initial parameterization has been performed. In the simplified representation, each protein was initially only represented by the position of its centre of mass and later with the inclusion of its orientation. The membrane protein used throughout most of this work was the bacterial outer membrane protein NanC, a member of the KdgM family of proteins. To parameterize the motion and interaction of proteins using MD, the potential of mean force (PMF) for the pairwise association of two proteins in a bilayer was calculated for a variety of orientational combinations, using a modified umbrella sampling procedure. The relative orientations chosen represented extreme examples of the contact regimes between the two proteins: they approximately corresponded to maxima and minima of the solvent inaccessible surface area, calculated when the proteins were in contact. These PMFs showed that there was a correlation between the buried surface area and the depth of the potential well in the PMF; this is something that, to date, has only been observed in these relatively-'featureless' membrane proteins (but is seen in globular proteins), where the effect of the interactions with lipids in the bilayer plays a larger role. Features in the PMF were observed that resulted from the preferential organization of lipids in the region between the two proteins. These features were small wells in the PMF, which occurred at protein separations that corresponded to the intervening lipids being optimally packed between the proteins. This result further highlighted the role that the lipids in the bilayer played in the interaction between the NanC proteins. The simplified bilayer model was parameterized using the PMFs and the relationship between buried surface area and potential well depth. The initial model included only the proteins' positions. A series of Monte Carlo simulations were performed in order to compare the system behaviour to that of an equivalent MD simulation. Initially, the MD simulation and our parameterized model did not show a good agreement, so a Monte Carlo scheme that incorporated cluster-based movements was implemented. The agreement between the MD simulation and the simulations of our model using the cluster-based scheme, when comparing diffusive and clustering behaviour, was good. Including the orientation-dependent features of the parameterization resulted in the emergence of behaviour that was not clearly detectable in the MD simulation. Finally, attempts were made to parameterize the model using PMFs for the association of rhodopsin from the literature. Rhodopsin was a much more complicated protein to represent: there was not a clear correlation between surface area and the features of the PMF, and the geometry of the interaction between two rhodopsins was more complicated. Simulations of the 'rows-of-dimers' system of rhodopsin, observed in disc membranes, was not entirely well represented by the model; for such a closely packed system, where the number of lipids is much closer to the number of proteins, the use of an implicit-lipid model meant that the effect of the reduced lipid mobility was not adequately captured. However, the model accurately captures the orientational composition of the system. Future work should be focussed on incorporating explicit representations of the lipid in the system so that the behaviour of close-packed systems are better represented.

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4

Dutta, Priyanka. "Computational Modeling of Allosteric Stimulation of Nipah Virus Host Binding Protein." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6227.

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Nipah belongs to the family of paramyxoviruses that cause numerous fatal diseases in humans and farm animals. There are no FDA approved drugs for Nipah or any of the paramyxoviruses. Designing antiviral therapies that are more resistant to viral mutations require understanding of molecular details underlying infection. This dissertation focuses on obtaining molecular insights into the very first step of infection by Nipah. Such details, in fact, remain unknown for all paramyxoviruses. Infection begins with the allosteric stimulation of Nipah virus host binding protein by host cell receptors. Understanding molecular details of this stimulation process have been challenging mainly because, just as in many eukaryotic proteins, including GPCRs, PDZ domains and T-cell receptors, host receptors induce only minor structural changes (< 2 Å) and, consequently, thermal fluctuations or dynamics play a key role. This work utilizes a powerful molecular dynamics based approach, which yields information on both structure and dynamics, laying the foundation for its future applications to other paramyxoviruses. It proposes a new model for the initial phase of stimulation of Nipah’s host binding protein, and in general, highlights that (a) interfacial waters can play a crucial role in the inception and propagation of allosteric signals; (b) extensive inter-domain rearrangements can be triggered by minor changes in the structures of individual domains; and (c) mutations in dynamically stimulated proteins can induce non-local changes that spread across entire domains.

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5

Parra, Katherine Cristina. "Combination of the Computational Methods: Molecular dynamics, Homology Modeling and Docking to Design Novel Inhibitors and study Structural Changes in Target Proteins for Current Diseases." Scholar Commons, 2014. https://scholarcommons.usf.edu/etd/5093.

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In this thesis, molecular dynamics simulations, molecular docking, and homology modeling methods have been used in combination to design possible inhibitors as well as to study the structural changes and function of target proteins related to diseases that today are in the spotlight of drug discovery. The inwardly rectifying potassium (Kir) channels constitute the first target in this study; they are involved in cardiac problems. On the other hand, tensin, a promising target in cancer research, is the second target studied here. The first chapter includes a brief update on computational methods and the current proposal of the combination of MD simulations and docking techniques, a procedure that is applied for the engineering of a new blocker for Kir2.1 ion channels and for the design of possible inhibitors for Tensin. Chapter two focuses in Kir ion channels that belong to the family of potassium-selective ion channels which have a wide range of physiological activity. The resolved crystal structure of a eukaryotic Kir channel was used as a secondary structure template to build the Kir-channels whose crystallographic structures are unavailable. Tertiapin (TPN), a 21 a.a. peptide toxin found in honey bee venom that blocks a type of Kir channels with high affinity was also used to design new Kir channel blockers. The computational methods homology modeling and protein-protein docking were employed to yield Kir channel-TPN complexes that showed good binding affinity scores for TPN-sensitive Kir channels, and less favorable for Kir channels insensitive to TPN block. The binding pocket of the insensitive Kir-channels was studied to engineer novel TPN-based peptides that show favorable binding scores via thermodynamic mutant-cycle analysis. Chapter three is focused on the building of homology models for Tensin 1, 2 and 3 domains C2 and PTP using the PTEN X-ray crystallographic structure as a secondary structure template. Molecular docking was employed for the screening of druggable small molecules and molecular dynamics simulations were also used to study the tensin structure and function in order to give some new insights of structural data for experimental binding and enzymatic assays. Chapter four describes the conformational changes of FixL, a protein of bradyrhizobia japonicum. FixL is a dimer known as oxygen sensor that is involved in the nitrogen fixation process of root plants regulating the expression of genes. Ligand behavior has been investigated after the dissociation event, also the structural changes that are involved in the relaxation to the deoxy state. Molecular dynamics simulations of the CO-bound and CO-unbound bjFixL heme domain were performed during 10 ns in crystal and solution environments then analyzed using Principal Component Analysis (PCA). Our results show that the diffusion of the ligand is influenced by internal motions of the bound structure of the protein before CO dissociation, implying an important role for Arg220. In turn, the location of the ligand after dissociation affects the conformational changes within the protein. The study suggests the presence of a cavity close to the methine bridge C of the heme group in agreement with spectroscopic probes and that Arg220 acts as a gate of the heme cavity.

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6

Guinto, Ferdiemar Cardenas Jr. "Investigating Secondary Structure Features of YAP1 Protein Fragments Using Molecular Dynamics (MD) and Steered Molecular Dynamics (SMD) Simulations." Scholarly Commons, 2017. https://scholarlycommons.pacific.edu/uop_etds/2973.

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Molecular dynamics (MD) is a powerful tool that can be applied to protein folding and protein structure. MD allows for the calculation of movement, and final position, of atoms in a biomolecule. These movements can be used to investigate the pathways that allow proteins to fold into energetically favorable structures. While MD is very useful, it still has its limitations. Most notable, computing power and time are of constant concern. Protein structure is inherently important due to the direct link between the structure of a protein and its function. One of the four levels of protein structure, the secondary structure, is the first level to accommodate for the three-dimensional shape of a protein. The main driving force behind secondary structure is hydrogen bonding, which occurs between the carboxyl oxygen and the amine hydrogen of the backbone of a peptide. Determining a greater link between hydrogen bond patterns and types of secondary structure can provide more insight on how proteins fold. Because molecular dynamics allows for an atomic level view of the dynamics behind protein folding/unfolding, it becomes very useful in observing the effects of particular hydrogen bond patterns on the folding pathway and final structure formed of a protein. Using molecular dynamic simulations, a series of experiments in an attempt to alter structure, hydrogen bonding, and folding patterns, can be performed. This information can be used to better understand the driving force of secondary structure, and use the knowledge gained to manipulate these simulations to force folding events, and with that, desired secondary structure features.

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7

Lumb, Craig Nicholas. "Computational studies of signalling at the cell membrane." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:d5b2db00-1050-4191-8eff-3521a4885a0c.

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In order to associate with the cytoplasmic leaflet of the plasma membrane, many cytosolic signalling proteins possess a distinct lipid binding domain as part of their overall fold. Here, a multiscale simulation approach has been used to investigate three membrane-binding proteins involved in cellular processes such as growth and proliferation. The pleckstrin homology (PH) domain from the general receptor for phosphoinositides 1 (GRP1-PH) binds phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃) with high affinity and specificity. To investigate how this peripheral protein is able to locate its target lipid in the complex membrane environment, Brownian dynamics (BD) simulations were employed to explore association pathways for GRP1-PH binding to PI(3,4,5)P₃ embedded in membranes with different surface charge densities and distributions. The results indicated that non-PI(3,4,5)P₃ lipids can act as decoys to disrupt PI(3,4,5)P₃ binding, but that at approximately physiological anionic lipid concentrations steering towards PI(3,4,5)P₃ is actually enhanced. Atomistic molecular dynamics (MD) simulations revealed substantial membrane penetration of membrane-bound GRP1-PH, evident when non-equilibrium, steered MD simulations were used to forcibly dissociate the protein from the membrane surface. Atomistic and coarse grained (CG) MD simulations of the phosphatase and tensin homologue deleted on chromosome ten (PTEN) tumour suppressor, which also binds PI(3,4,5)P₃, detected numerous non-specific protein-lipid contacts and anionic lipid clustering around PTEN that can be modulated by selective in silico mutagenesis. These results suggested a dual recognition model of membrane binding, with non-specific membrane interactions complementing the protein-ligand interaction. Molecular docking and MD simulations were used to characterise the lipid binding properties of kindlin-1 PH. Simulations demonstrated that a dynamic salt bridge was responsible for controlling the accessibility of the binding site. Electrostatics calculations applied to a variety of PH domains suggested that their molecular dipole moments are typically aligned with their ligand binding sites, which has implications for steering and ligand electrostatic funnelling.

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8

Pavlovicz, Ryan Elliott. "Investigation of Protein/Ligand Interactions Relating Structural Dynamics to Function: Combined Computational and Experimental Approaches." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397220613.

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9

Cardoch, Sebastian. "Computational study of single protein sensing using nanopores." Thesis, Uppsala universitet, Materialteori, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-423441.

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Identifying the protein content in a cell in a fast and reliable manner has become a relevant goal in the field of proteomics. This thesis computationally explores the potential for silicon nitride nanopores to sense and distinguish single miniproteins, which are small domains that promise to facilitate the systematic study of larger proteins. Sensing and identification of these biomolecules using nanopores happens by studying modulations in ionic current during translocation. The approach taken in this work was to study two miniproteins of similar geometry, using a cylindrical-shaped pore. I employed molecular mechanics to compare occupied pore currents computed based on the trajectory of ions. I further used density functional theory along with relative surface accessibility values to compute changes in interaction energies for single amino acids and obtain relative dwell times. While the protein remained inside the nanopore, I found no noticeable differences in the occupied pore currents of the two miniproteins for systems subject to 0.5 and 1.0 V bias voltages. Dwell times were estimated based on the translocation time of a protein that exhibits no interaction with the pore walls. I found that both miniproteins feel an attractive force to the pore wall and estimated their relative dwell times to differ by one order of magnitude. This means even in cases where two miniproteins are indistinguishable by magnitude changes in the ionic current, the dwell time might still be used to identify them. This work was an initial investigation that can be further developed to increase the accuracy of the results and be expanded to assess other miniproteins with the goal to aid future experimental work.

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10

Zhou, Guangfeng. "STATISTICAL MODELS AND THEIR APPLICATIONS IN STUDYING BIOMOLECULAR CONFORMATIONAL DYNAMICS." Diss., Temple University Libraries, 2017. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/478773.

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Chemistry
Ph.D.
It remains a major challenge in biophysics to understand the conformational dynamics of biomolecules. As powerful tools, molecular dynamics (MD) simulations have become increasingly important in studying the full atomic details of conformational dynamics of biomolecules. In addition, many statistical models have been developed to give insight into the big datasets from MD simulations. In this work, I first describe three statistical models used to analyze MD simulation data: Lifson-Roig Helix-Coil theory, Bayesian inference models, and Markov state models. Then I present the applications of each model in analyzing MD simulations and revealing insight into the conformational dynamics of biomolecules. These statistical models allow us to bridge microscopic and macroscopic mechanisms of biological processes and connect simulations with experiments.
Temple University--Theses

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11

Buch, Mundó Ignasi 1984. "Investigation of protein-ligand interactions using high-throughput all-atom molecular dynamics simulations." Doctoral thesis, Universitat Pompeu Fabra, 2012. http://hdl.handle.net/10803/101407.

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Investigation of protein-ligand interactions has been a long-standing application for molecular dynamics (MD) simulations given its importance to drug design. However, relevant timescales for biomolecular motions are orders of magnitude longer than the commonly accessed simulation times. Adequate sampling of biomolecular phase-space has therefore been a major challenge in computational modeling that has limited its applicability. The primary objective for this thesis has been the brute-force simulation of costly protein-ligand binding modeling experiments on a large computing infrastructure. We have built and developed GPUGRID: a peta-scale distributed computing infrastructure for high-throughput MD simulations. We have used GPUGRID for the calculation of protein-ligand binding free energies as well as for the reconstruction of binding processes through unguided ligand binding simulations. The promising results presented herein, may have set the grounds for future applications of high-throughput MD simulations to drug discovery programs.

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12

Rajapaksha, Suneth P. "Single Molecule Spectroscopy Studies of Membrane Protein Dynamics and Energetics by Combined Experimental and Computational Analyses." Bowling Green State University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1337141955.

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13

Dahl, Anna Caroline E. "Membrane protein mechanotransduction : computational studies and analytics development." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:67798647-8ed5-46e0-bde9-c71235fe70ba.

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Membrane protein mechanotransduction is the altered function of an integral membrane protein in response to mechanical force. Such mechanosensors are found in all kingdoms of life, and increasing numbers of membrane proteins have been found to exhibit mechanosensitivity. How they mechanotransduce is an active research area and the topic of this thesis. The methodology employed is classical molecular dynamics (MD) simulations. MD systems are complex, and two programs were developed to reduce this apparent complexity in terms of both visual abstraction and statistical analysis. Bendix detects and visualises helices as cylinders that follow the helix axis, and quantifies helix distortion. The functionality of Bendix is demonstrated on the symporter Mhp1, where a state is identified that had hitherto only been proposed. InterQuant tracks, categorises and orders proximity between parts of an MD system. Results from multiple systems are statistically interrogated for reproducibility and significant differences at the resolution of protein chains, residues or atoms. Using these tools, the interaction between membrane and the Escherichia coli mechanosensitive channel of small conductance, MscS, is investigated. Results are presented for crystal structures captured in different states, one of which features electron density proposed to be lipid. MD results supports this hypothesis, and identify differential lipid interaction between closed and open states. It is concluded that propensity for lipid to leave for membrane bulk drives MscS state stability. In a subsequent study, MscS is opened by membrane surface tension for the first time in an MD setup. The gating mechanism of MscS is explored in terms of both membrane and protein deformation in response to membrane stretch. Using novel tension methodology and the longest MD simulations of MscS performed to date, a molecular basis for the Dashpot gating mechanism is proposed. Lipid emerges as an active structural element with the capacity to augment protein structure in the protein structure-function paradigm.

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14

Schmidt, Matthias Rene. "K+ channels : gating mechanisms and lipid interactions." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:51dc4149-d943-4dcd-bf5b-f04130456d84.

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Computational methods, including homology modelling, in-silico dockings, and molecular dynamics simulations have been used to study the functional dynamics and interactions of K⁺ channels. Molecular models were built of the inwardly rectifying K⁺ channel Kir2.2, the bacterial homolog K⁺ channel KirBac3.1, and the twin pore (K2P) K⁺ channels TREK-1 and TRESK. To investigate the electrostatic energy profile of K⁺ permeating through these homology models, continuum electrostatic calculations were performed. The primary mechanism of KirBac3.1 gating is believed to involve an opening at the helix bundle crossing (HBC). However, simulations of Kir channels have not yet revealed opening at the HBC. Here, in simulations of the new KirBac3.1-S129R X-ray crystal structure, in which the HBC was trapped open by the S129R mutation in the inner pore-lining helix (TM2), the HBC was found to exhibit considerable mobility. In a simulation of the new KirBac3.1-S129R-S205L double mutant structure, if the S129R and the S205L mutations were converted back to the wild-type serine, the HBC would close faster than in the simulations of the KirBac3.1-S129R single mutant structure. The double mutant structure KirBac3.1-S129R-S205L therefore likely represents a higher-energy state than the single mutant KirBac3.1-S129R structure, and these simulations indicate a staged pathway of gating in KirBac channels. Molecular modelling and MD simulations of the Kir2.2 channel structure demonstrated that the HBC would tend to open if the C-linker between the transmembrane and cytoplasmic domain was modelled helical. The electrostatic energy barrier for K⁺ permeation at the helix bundle crossing was found to be sensitive to subtle structural changes in the C-linker. Charge neutralization or charge reversal of the PIP2-binding residue R186 on the C-linker decreased the electrostatic barrier for K⁺ permeation through the HBC, suggesting an electrostatic contribution to the PIP2-dependent gating mechanism. Multi-scale simulations determined the PIP2 binding site in Kir2.2, in good agreement with crystallographic predictions. A TREK-1 homology model was built, based on the TRAAK structure. Two PIP2 binding sites were found in this TREK-1 model, at the C-terminal end, in line with existing functional data, and between transmembrane helices TM2 and TM3. The TM2-TM3 site is in reasonably good agreement with electron density attributed to an acyl tail in a recently deposited TREK-2 structure.

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15

Pham, Khoa Ngoc. "Conformational Dynamics and Stability Associated with Magnesium or Calcium Binding to DREAM in the Regulation of Interactions between DREAM and DNA or Presenilins." FIU Digital Commons, 2016. http://digitalcommons.fiu.edu/etd/2589.

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Downstream regulatory element antagonist modulator (DREAM) is involved in various interactions with targets both inside and outside of the nucleus. In the cytoplasm, DREAM interacts with the C-terminal fragments of presenilins to facilitate the production of β-amyloid plaques in Alzheimer’s disease. In the nucleus, Ca2+ free DREAM directly binds to specific downstream regulatory elements of prodynorphin/c-fos gene to repress the gene transcription in pain modulation. These interactions are regulated by Ca2+ and/or Mg2+ association at the EF-hands in DREAM. Therefore, understanding the conformational dynamics and stability associated with Ca2+ and/or Mg2+ binding to DREAM is crucial for elucidating the mechanisms of interactions of DREAM with DNA or presenilins. The critical barrier for envisioning the mechanisms of these interactions lies in the lack of NMR/crystal structures of Apo and Mg2+DREAM. Using a combination of fluorescence spectroscopy, circular dichroism, isothermal titration calorimetry, photothermal spectroscopy, and computational approaches, I showed that Mg2+ association at the EF-hand 2 structurally stabilizes the N-terminal alpha-helices 1, 2, and 5, facilitating the interaction with DNA. Binding of Ca2+ at the EF-hand 3 induces significant structural changes in DREAM, mediated by several hydrophobic residues in both the N- and C-domains. These findings illustrate the critical role of EF-hand 3 for Ca2+ signal transduction from the C- to N-terminus in DREAM. The Ca2+ association at the EF-hand 4 stabilizes the C-terminus by forming a cluster consisting of several hydrophobic residues in C-terminal domain. I also demonstrated that association of presenilin-1 carboxyl peptide with DREAM is Ca2+ dependent and the complex is stabilized by aromatic residues F462 and F465 from presenilin-1 and F252 from DREAM. Stabilization is also provided by residues R200 and R207 in the loop connecting a7 and a8 in DREAM and the residues D450 and D458 in presenilin-1. These findings provide a structural basis for the development of new drugs for chronic pain and Alzheimer’s disease treatments.

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16

Chakraborty, Srirupa. "Computational modeling of structural dynamics and energetics of two allosteric proteins| Kinesins and Acetylcholine Receptors." Thesis, State University of New York at Buffalo, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10242471.

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To quote famous physicist and Nobel laureate, Dr. Richard Feynman, “…everything that living things do can be understood in terms of the jigglings and wigglings of atoms.” It is these jigglings and wigglings of atoms that form the focus of my dissertation, which studies the structural dynamics of two different allosteric proteins through computational simulations. Protein allostery is a field that has been investigated widely. But the structural details of how signals initiating at one site transmit through the network of residues in different proteins and result in the alteration of their functional states, still remains largely unresolved. Here, we independently study the kinesin motor protein and the neuromuscular acetylcholine receptor (nAChR) – both of which play crucial roles in cellular signaling. Kinesins are intracellular porters, carrying organelles, molecules and other cargo within the cell, while nAChRs are transmembrane receptors that aid in intercellular communication at nerve-to-muscle synapses. These two protein families are structurally and functionally very different, but both are allosteric in nature, with interesting protein dynamics that efficiently convert chemical energy to mechanical motions in order to perform their cellular functions.

The total timescale of an entire allosteric transition is currently too long for complete all-atom molecular dynamics simulations. Thus, in this dissertation, for both the projects, we begin at different equilibrium states of the proteins and carry out comparative analyses of conformation and dynamics at those states, which aids in elucidating the structural and functional correlates for these systems.

For the kinesin-microtubule (KIN-MT) system, we have built atomistic structure models for the key nucleotide-binding states of the kinesin-MT complex from lower resolution cryo-EM maps, by suitably modifying the MD potential with the EM map force. We have also studied ligand-protein (ADP/ATP-kinesin) interactions and predicted the sequence of structural changes in kinesin-MT complex during its conformational transitions between important biochemical states and pinpointed key contributing residues.

Simultaneously, we have also characterized the transmitter binding sites of neuromuscular acetylcholine receptors and analyzed the energy asymmetries between the fetal and adult endplate receptors. Through large-scale simulations of the fetal and adult binding sites, we have come across compelling evidence of the structural causes that explain these asymmetries and were successful in identifying the minimum construct that is both necessary and sufficient to exchange the function between adult and fetal binding sites in AChRs. Our in silico models and predictions act as important tools to further guide mutational and functional experiments.

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17

Abd, Halim Khairul Bariyyah. "Molecular dynamics simulation studies of transmembrane signalling proteins." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:bc9e1e0e-433c-4adb-8374-1065eac0f37e.

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Receptor tyrosine kinases (RTKs) are a major class of cell surface receptors, important in cell signalling events associated with a variety of functions. High-throughput (HTP), coarse-grained molecular dynamics (CG-MD) simulations have been used to investigate the dimerization of the transmembrane (TM) domain of selected RTKs, including epidermal growth factor receptor (EGFR) and muscle-specific kinase (MuSK). EGFR activation requires not only a specific TM dimer interface, but also a proper orientation of its juxtamembrane (JM) domain. Phosphatidylinositol 4,5-bisphosphate (PIP₂) is known to abolish EGFR phosphorylation through interaction with basic residues within the JM domain. Here, a multiscale approach was used to investigate anionic lipid clustering around the TM-JM junction and how such clustering is modulated by the mutation of basic residues. The simulations demonstrated that PIP₂ may help stabilize the JM-A antiparallel dimer, which may in turn help stabilize TM domain helix packing of the N-terminal dimerization motif. A proximal TM domain residue has been implicated in the inhibition of ganglioside GM3 in phase-separated membranes. Here, CG simulations were used to explore the dynamic behaviour of the EGFR TM domain dimer in GM3-containing and GM3-depleted bilayers designed to resemble lipid-disordered (Ld) and phase-separated (Ld/Lo) membranes. The simulations suggest that the presence of GM3 in Ld/Lo bilayers can disrupt and destabilize the TM dimer, which helps to explain why GM3 may favour monomeric EGFR in vivo. To gain insights into the dynamic nature of the intact EGFR, a nearly complete EGFR dimer was modelled using available structural data and embedded in an asymmetric compositional complex bilayer, which resembles the mammalian plasma membrane. The results demonstrated the dynamic nature of the EGFR ectodomain and its predicted interactions with lipids in the local bilayer. Strong protein-lipid interactions, as well as lipid-lipid interactions, affect the local clustering of lipids and the diffusion of lipids in the vicinity of the protein on both leaflets.

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18

Mohammadiarani, Hossein. "Simulation Studies of Signaling and Regulatory Proteins." Thesis, University of New Hampshire, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10685640.

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I used molecular dynamics (MD) simulations as a primary tool to study folding and dynamics of signaling and regulatory proteins. Specifically, I have studied two classes of proteins: the first part of my thesis reports studies on peptides and receptors of the insulin family, and the second part reports on studies of regulatory proteins from the G-protein coupled receptor family. The first problem that I investigated was understanding the folding mechanism of the insulin B-chain and its mimetic peptide (S371) which were studied using enhanced sampling simulation methods. I validated our simulation approaches by predicting the known solution structure of the insulin B-chain helix and then applied them to study the folding of the mimetic peptide S371. Potentials of mean force (PMFs) along the reaction coordinate for each peptide are further resolved using the metadynamics method. I further proposed receptor-bound models of S371 that provide mechanistic explanations for competing binding properties of S371 and a tandem hormone-binding element of the receptor known as the C-terminal (CT) peptide. Next, I studied the all-atom structural models of peptides containing 51 residues from the transmembrane regions of IR and the type-1 insulin-like growth factor receptor (IGF1R) in a lipid membrane. In these models, the transmembrane regions of both receptors adopt helical conformations with kinks at Pro961 (IR) and Pro941 (IGF1R), but the C-terminal residues corresponding to the juxta-membrane region of each receptor adopt unfolded and flexible conformations in IR as opposed to a helix in IGF1R. I also observe that the N-terminal residues in IR form a kinked-helix sitting at the membrane-solvent interface, while homologous residues in IGF1R are unfolded and flexible. These conformational differences result in a larger tilt-angle of the membrane-embedded helix in IGF1R in comparison to IR to compensate for interactions with water molecules at the membrane-solvent interfaces. The metastable/stable states for the transmembrane domain of IR, observed in a lipid bilayer, are consistent with a known NMR structure of this domain determined in detergent micelles, and similar states in IGF1R are consistent with a previously reported model of the dimerized transmembrane domains of IGF1R. I further studied dimerization propensities of IR transmembrane domains using three different constructs in a lipid bilayer (isolated helices, ectodomain-anchored helices, and kinase-anchored helices). These studies revealed that the transmembrane domains can dimerize in isolation and in kinase-anchored forms, but not significantly in the ectodomain construct. The final studies in my thesis are focused on interplay of protein dynamics and small-molecule inhibition in a set of regulatory proteins known as the Regulators of G-protein Signaling (RGS) proteins. Thiadiazolidinone (TDZD) compounds have been shown to inhibit the protein-protein interaction between RGS and the alpha subunit of G-proteins by covalent modification of cysteine residues in RGS proteins. However, some of these cysteines in RGS proteins are not surface-exposed. I hypothesized that transient binding pockets expose cysteine residues differentially between different RGS isoforms. To explore this hypothesis, long time-scale classical MD simulations were used to probe the dynamics of three RGS proteins (RGS4, RGS8, and RGS19), and characterize flexibility in various helical motifs. The results from simulation studies were validated by hydrogen-deuterium exchange (HDX) studies, and revealed motions indicating solvent exposure of buried cysteine residues, thereby providing insights into inhibitor binding mechanisms. In addition, I used different published HDX models which have resulted in a comprehensive comparison of existing models. Furthermore, I developed the new HDX models with optimized parameters which had comparable accuracy and more computational efficiency compared to other models. Overall, my thesis has resulted in the development and applications of several state-of-the-art computational methods that have provided a detailed mechanistic understanding of peptide and small-molecule based inhibitors and their interactions with large proteins that are potentially useful in designing novel approaches to target protein-protein interactions.

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19

Ainsley, Jon. "Computational simulations of enzyme dynamics and the modelling of their reaction mechanisms." Thesis, Northumbria University, 2017. http://nrl.northumbria.ac.uk/36286/.

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Proteins and enzymes are large and complex biological molecules, characterized by unique three-dimensional structure are highly flexible and dynamic nature. Thorough understanding of protein and enzyme function requires studying of their conformational flexibility, because important physiological processes, such as ligand binding and catalysis rely on an enzyme’s dynamic nature and their ability to adopt a variety of conformational states. Computational methods are widely applied in studying enzymes and proteins structure and function providing a detailed atomistic-level of resolution data about the dynamics and catalytic processes, mechanisms in biomolecules, therefore even more nowadays a term ‘computational enzymology’ has emerged. Experimental methods often have difficulty in predicting dynamic motions of proteins. Computational simulations techniques, such as Molecular Dynamics simulations, have proven successful in simulating the conformational flexibility of proteins in studying structure-function relationships. Additionally, the binding events between two molecules, e.g. an enzyme and its substrate, can be computationally predicted with molecular docking methods. Enzymes are proteins that catalyse almost all biochemical reactions and metabolic processes in all organisms. In order to study the conformational flexibility of proteins we apply molecular dynamics simulations, and in order to simulate their reaction mechanisms we apply quantum mechanical simulations. Quantum mechanical simulations can also be used to predict the electronic structure of organic compounds, by calculating their electronic structures we perform orbital analyses and predict their optical properties. The results gained from our computational simulations can give new insights into explanation of experimental findings and data and can inspire and guide further experiments.

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20

Shorthouse, David Robert. "Computational methods for the study of immunoglobulin aggregation." Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:43c0950e-7f58-48b9-899d-74dcfee35887.

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Protein aggregation is a major challenge in the development of antibody-based therapeutics. Therapeutic antibodies are produced and stored in high concentrations and under fluctuating conditions unfavourable for their stability. Aggregation of these proteins in solution leads to serious consequences for patients, with the initiation of immune reactions, which have the potential to be fatal, and in the loss of clinical potency. The types of aggregates formed by antibodies, and the processes that lead to their propagation are poorly understood. By studying these molecules via computational approaches, we are able to simulate and probe their tendency to aggregate on experimentally comparable timescales. By performing small numbers of coarse grained simulations of immunoglobulin frag- ments it is shown that specific regions of proteins are involved in self-self interactions, and these regions are targets for reducing the self-association of experimental molecules. Techniques developed here are integrated within a high throughput approach that is able to generate information on aggregation for a large number of candidate antibody structures. The methodology was refined via development of a novel technique for coarse grained simulations of oligosaccharides. This method was initially tested on glycolipids, and then extended to glycoproteins. The primary outcome is a coarse grained model for a glyco- sylated antibody Fc fragment. The glycosylated Fc was then simulated, and compared to experimental data. Coarse grained simulations support the hypothesis that the protein be- comes more flexible in the absence of glycosylation.

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21

Krammer, André Thomas. "Computational studies of protein-membrane interactions and forced unfolding of proteins /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/9697.

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22

Karolak, Aleksandra. "Application and Development of Computational Methods in Conformational Studies of Bio-molecules." Scholar Commons, 2015. https://scholarcommons.usf.edu/etd/5520.

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The work presented in my dissertation focuses on the conformational studies of bio-molecules including proteins and DNA using computational approaches. Conformational changes are important in numerous molecular bioprocesses such as recognition, transcription, replication and repair, etc. Proteins recognize specific DNA sequences and upon binding undergo partial or complete folding or partial unfolding in order to find the optimal conformational fit between molecules involved in the complex. In addition to sequence specific recognition, proteins are able to distinguish between subtle differences in local geometry and flexibility associated with DNA that may further affect their binding affinities. Experimental techniques provide high-resolution details to the static structures but the structural dynamics are often not accessible with these methods; but can be probed using computational tools. Various well-established molecular dynamics methods are used in this work to study differences in geometry and mechanical properties of specific systems under unmodified and modified conditions. Briefly, the studies of several protein and DNA systems investigated the importance of local interactions and modifications for the stability, geometry and mechanical properties using standard and enhanced molecular dynamics simulations. In addition to the conformational studies, the development of a new method for enhanced sampling of DNA step parameters and its application to DNA systems is discussed. Chapter 1 reviews the importance of the conformational changes in bioprocesses and the theory behind the computational methods used in this work. In the project presented in chapter 2 unbiased molecular dynamics and replica exchange molecular dynamics are employed to identify the specific local contacts within the inhibitory module of ETS-1. ETS-1 is a human transcription factor important for normal but also malignant cell growth. An increased concentration of this protein is related to a negative prognosis in many cancers. A part of the inhibitory module, inhibitory helix 1 (HI-1) is located on the site of the protein opposite to the DNA binding site and although loosely packed, stays folded in the apo state and unfolds upon ETS-1 binding to DNA. Our study investigated the character and importance of contacts between HI-1 and neighboring helices of the inhibitory module: HI-2 and H4. We also identified a mutant of HI-1, which possessed the higher helical propensity than the original construct. This study supported the experimental findings and enhanced the field by the identification of new potential target for experimental tests of the system, which plausibly inhibits binding to DNA. In the studies discussed in chapters 3-5 the conformational dynamics of DNA under normal conditions and upon specific epigenetic modifications are presented. Since DNA conformation can be accurately described by six base pair step parameters: twist, tilt, roll, shift, slide and rise, these were extensively analyzed and the results elucidated insights into the properties of the systems. In order to enhance unbiased simulations and allow for easier crossing of the energy barriers, we developed and implemented a novel method to control DNA base pair step parameters. With this approach we obtained the free energy estimates of e.g. DNA rearrangements in a more efficient manner. This advanced computational method, supported by standard and additional enhanced techniques, was then applied in the studies of DNA methylation on cytosine or adenine bases and oxidative damage of cytosine.

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23

Tangar, Antonija. "Structure-Function Relationships in Hexacoordinate Heme Proteins: Mechanism of Cytoglobin Interactions with Exogenous Ligands." FIU Digital Commons, 2018. https://digitalcommons.fiu.edu/etd/3729.

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Cytoglobin (Cygb) and neuroglobin (Ngb) are among the newest members of vertebrate globin family characterized by a classical 3-over-3 α-helical fold and a heme prosthetic group capable of reversibly binding small ligands such as O2, CO and NO. The physiological functions of Cygb and Ngb remain to be determined; however, current data suggest that both proteins have a significant role in cytoprotection in hypoxic and genotoxic conditions. Cytoglobin and Ngb are distinct from their better-known counterparts, hemoglobin (Hb) and myoglobin (Mb), in several structural features. First, in the absence of an external ligand, the sixth coordination site of the heme iron in Cygb and Ngb is occupied by a distal histidine residue, leading to a complex ligand rebinding mechanism dependent on the rate of distal His dissociation from the heme iron. Although hexacoordination was observed before in plant and bacterial hemoglobins, the physiological role of this feature remains unknown. Second, both Ngb and Cygb are capable of forming an intraprotein disulfide bond, which has been shown to regulate ligand binding affinity, leading to a hypothesis that intracellular function of these proteins is redox-dependent. Lastly, Cygb contains 20 amino acid long extensions on both N- and C- termini, a unique feature among vertebrate globins with unknown physiological function. The work presented in the dissertation reveals that hexacoordinate heme reactivity is distinct from that of pentacoordinate heme and is strongly influenced by the distal histidine residue and the disulfide bond. In the case of human Cygb, experimental and computational approaches demonstrated that the disulfide bond regulates the flexibility of the N terminus and the accessibility of the 1,8-ANS binding site. Furthermore, molecular dynamics of the hexa- and pentacoordinate human Ngb were probed computationally to elucidate structural requirements that govern signal transmission between CD loop and the distal pocket. Lastly, Ngb and Cygb were reconstituted with a fluorescent analog of the native heme group to produce hexacoordinate variants with favorable photophysical properties that can be used to characterize protein-protein interactions.

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24

Westerlund, Annie M. "Computational Study of Calmodulin’s Ca2+-dependent Conformational Ensembles." Licentiate thesis, KTH, Biofysik, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-234888.

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Ca2+ and calmodulin play important roles in many physiologically crucial pathways. The conformational landscape of calmodulin is intriguing. Conformational changes allow for binding target-proteins, while binding Ca2+ yields population shifts within the landscape. Thus, target-proteins become Ca2+-sensitive upon calmodulin binding. Calmodulin regulates more than 300 target-proteins, and mutations are linked to lethal disorders. The mechanisms underlying Ca2+ and target-protein binding are complex and pose interesting questions. Such questions are typically addressed with experiments which fail to provide simultaneous molecular and dynamics insights. In this thesis, questions on binding mechanisms are probed with molecular dynamics simulations together with tailored unsupervised learning and data analysis. In Paper 1, a free energy landscape estimator based on Gaussian mixture models with cross-validation was developed and used to evaluate the efficiency of regular molecular dynamics compared to temperature-enhanced molecular dynamics. This comparison revealed interesting properties of the free energy landscapes, highlighting different behaviors of the Ca2+-bound and unbound calmodulin conformational ensembles. In Paper 2, spectral clustering was used to shed light on Ca2+ and target protein binding. With these tools, it was possible to characterize differences in target-protein binding depending on Ca2+-state as well as N-terminal or C-terminal lobe binding. This work invites data-driven analysis into the field of biomolecule molecular dynamics, provides further insight into calmodulin’s Ca2+ and targetprotein binding, and serves as a stepping-stone towards a complete understanding of calmodulin’s Ca2+-dependent conformational ensembles.

QC 20180912

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25

Kotecha, Abhay. "Structure and dynamics of picornavirus capsids to inform vaccine design." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:df739a5f-fdb2-4930-909f-d94ce274ce33.

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The physical properties of viral capsids are major determinants of vaccine efficacy for several picornaviruses which impact on human and animal health. Current picornavirus vaccines are frequently produced from inactivated virus. Inactivation often reduces the stability of the virus capsid, causing a problem for Foot and Mouth Disease Virus (FMDV) where certain serotypes fall apart into pentameric assemblies below pH 6.5 or at temperatures slightly above 37°C, destroying their effectiveness in eliciting a protective immune response. As a result, vaccines require a cold chain for storage and animals need to be frequently immunised. FMDV is a member of the Aphthovirus genus of the Picornaviridae. Globally there are seven FMDV serotypes: O, A, Asia1, C and SAT-1, -2 and -3, contributing to a dynamic pool of antigenic variation. As part of collaboration between the Division of Structural Biology, Oxford University, The Pirbright Institute, Reading University and ARC, Ondespoort, South Africa we sought to rationally engineer thermo-stable FMDV capsids either as infectious copy virus or recombinant empty capsids with improved thermo-stability for improved vaccines. In this project, in silico molecular dynamics (MD) simulations, molecular modelling, free energy calculations, X-ray crystallography, electron microscopy and various biochemical/biophysical techniques were used to design and help characterise the capsids. For the most unstable FMDV serotypes (O and SAT2), panels of stabilising mutants were characterised by MD. Promising candidates were then engineered and shown to confer increased thermo- and pH-stability. Thus, in silico predictions translate into marked stabilisation of both infectious and recombinant empty viral capsids. A novel in situ method was used to determine crystal structures for quality assessment and to verify that no unanticipated structural changes have occurred as a consequence of the modifications made. The structures of the wildtype and two of the stabilised mutants were solved and the antigenic surfaces shown to be unchanged. Animal trials showed stabilised particles can generate a similar or improved neutralising antibody response compared to the traditional vaccines and may therefore lead to a new generation of stable and safe vaccines.

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26

Kognole, Abhishek A. "UNDERSTANDING CARBOHYDRATE RECOGNITION MECHANISMS IN NON-CATALYTIC PROTEINS THROUGH MOLECULAR SIMULATIONS." UKnowledge, 2018. https://uknowledge.uky.edu/cme_etds/80.

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Non-catalytic protein-carbohydrate interactions are an essential element of various biological events. This dissertation presents the work on understanding carbohydrate recognition mechanisms and their physical significance in two groups of non-catalytic proteins, also called lectins, which play key roles in major applications such as cellulosic biofuel production and drug delivery pathways. A computational approach using molecular modeling, molecular dynamic simulations and free energy calculations was used to study molecular-level protein-carbohydrate and protein-protein interactions. Various microorganisms like bacteria and fungi secret multi-modular enzymes to deconstruct cellulosic biomass into fermentable sugars. The carbohydrate binding modules (CBM) are non-catalytic domains of such enzymes that assist the catalytic domains to recognize the target substrate and keep it in proximity. Understanding the protein-carbohydrate recognition mechanisms by which CBMs selectively bind substrate is critical to development of enhanced biomass conversion technology. We focus on CBMs that target both oligomeric and non-crystalline cellulose while exhibiting various similarities and differences in binding specificity and structural properties; such CBMs are classified as Type B CBMs. We show that all six cellulose-specific Type B CBMs studied in this dissertation can recognize the cello-oligomeric ligands in bi-directional fashion, meaning there was no preference towards reducing or non-reducing end of ligand for the cleft/groove like binding sites. Out of the two sandwich and twisted forms of binding site architectures, twisted platform turned out to facilitate tighter binding also exhibiting longer binding sites. The exterior loops of such binding sites were specifically identified by modeling the CBMs with non-crystalline cellulose showing that high- and low-affinity binding site may arise based on orientation of CBM while interacting with non-crystalline substrate. These findings provide various insights that can be used for further understanding of tandem CBMs and for various CBM based biotechnological applications. The later part of this dissertation reports the identification of a physiological ligand for a mammalian glycoprotein YKL-40 that has been only known as a biomarker in various inflammatory diseases and cancers. It has been shown to bind to oligomers of chitin, but there is no known function of YKL-40, as chitin production in the human body has never been reported. Possible alternative ligands include proteoglycans, polysaccharides, and fibers such as collagen, all of which make up the mesh comprising the extracellular matrix. It is likely that YKL-40 is interacting with these alternative polysaccharides or proteins within the body, extending its function to cell biological roles such as mediating cellular receptors and cell adhesion and migration. We considered the feasibility of polysaccharides, including cello-oligosaccharides, hyaluronan, heparan sulfate, heparin, and chondroitin sulfate, and collagen-like peptides as physiological ligands for YKL-40. Our simulation results suggest that chitohexaose and hyaluronan preferentially bind to YKL-40 over collagen, and hyaluronan is likely the preferred physiological ligand, as the negatively charged hyaluronan shows enhanced affinity for YKL-40 over neutral chitohexaose. Collagen binds in two locations at the YKL-40 surface, potentially related to a role in fibrillar formation. Finally, heparin non- specifically binds at the YKL-40 surface, as predicted from structural studies. Overall, YKL-40 likely binds many natural ligands in vivo, but its concurrence with physical maladies may be related to the associated increases in hyaluronan.

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27

Chetwynd, Alan. "Computational studies of transmembrane helix insertion and association." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:440da098-5bd6-4fcb-8396-645517ac2122.

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Membrane proteins perform a variety of functions essential for the viability of the cell, including transport and signalling across the membrane. Most membrane proteins are formed from bundles of transmembrane helices. In this thesis molecular dynamics simulations have been used to investigate helix insertion into bilayers and helix association within bilayers. The potentials of mean force for the insertion of helices derived from the cystic ﬁbrosis transmembrane conductance regulator into lipid bilayers were calculated using coarse-grained molecular dynamics simulations. The results showed that the insertion free energy increased with helix length and bilayer hydrophobic width. The insertion free energies obtained were signiﬁcantly larger than comparable quantities obtained from translocon- mediated insertion experiments, consistent with a variety of previous studies. The implications of this observation for the interpretation of in vivo translocon-mediated insertion experiments, and the function of the translocon, are discussed. Coarse-grained and atomistic molecular dynamics simulations of the transmembrane region of the receptor tyrosine kinase EphA1 suggested that the transmembrane helix dimer was most stable when interacting via the glycine zipper motif, in agreement with a structure obtained by NMR spectroscopy. Coarse-grained simulations of the transmembrane region of EphA2 suggested that the dimer has two stable orientations, interacting via a glycine zipper or a heptad motif. Both structures showed right-handed dimers, although an NMR structure of the transmembrane region of EphA2 shows a left-handed dimer interacting via the heptad motif. Both structures obtained from coarse-grained simulations proved unstable when simulated at an atomistic level of detail. The potentials of mean force for dissociating the EphA1 and EphA2 dimers were calcu- lated using coarse-grained molecular dynamics calculations. Convergence of the detailed structure of the proﬁles was not conclusively shown, although association free energies cal- culated from the proﬁles were consistent over a variety of simulation times. The association free energies were slightly larger than experimental values obtained for comparable sys- tems, but consistent with similar computational calculations previously reported. However, direct comparisons are difﬁcult owing to the inﬂuence of environmental factors on reported association free energies. The potential of mean force proﬁles showed that the interaction via the glycine zipper motif for EphA1 was signiﬁcantly more stable than any other confor- mation. For EphA2 the potential of mean force proﬁles suggested that interaction via the glycine zipper and heptad motifs both provided stable or metastable conformations, with the interaction via the glycine zipper motif probably at least as stable as that via the heptad motif.

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Nadas, Janos Istvan. "Computational Structure Activity Relationship Studies on the CD1d/Glycolipid/TCR Complex using AMBER and AUTODOCK." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1251145085.

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29

Chotikasemsri, Pongsathorn. "Computational Prediction of the Agregated Structure of Denatured Lysozyme." TopSCHOLAR®, 2009. http://digitalcommons.wku.edu/theses/120.

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Mis-folded proteins and their associated aggregates are a contributing factor in some human diseases. In this study we used the protein lysozyme as a model to define aggregation structures under denaturing conditions. Sasahara et al. (2007), Frare et al. (2009, 2006), and Rubin et al. (2008) observed conditions where heat denatured lysozyme formed fibril structures that were observed to be 8-17 nanometers in diameter under the electron microscope. Even though the crystal structure of lysozyme is known, the denatured form of this protein is still unknown. Therefore, we used Rosetta++ protein folding and blind docking software to create in silico models of the protein at denaturing temperatures and subsequently docked them into aggregates. Here we compare those structures and select forms consistent with the fibril structure from the previous papers. The next step is to be able to use the predicted models of the fibrilar forms of denatured lysozyme to help us understand the exact conformation of fibril structures. This will let us confirm the docking interactions during the fibril aggregation process. The ultimate goal is to use the validated denatured structures to model interactions with heat shock proteins during the dis-aggregation process.

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30

Walker, Alice Rachel. "Computational Simulations of Cancer and Disease-Related Enzymatic Systems Using Molecular Dynamics and Combined Quantum Methods." Thesis, University of North Texas, 2018. https://digital.library.unt.edu/ark:/67531/metadc1157647/.

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This work discusses applications of computational simulations to enzymatic systems with a particular focus on the effects of various small perturbations on cancer and disease-related systems. First, we cover the development of carbohydrate-based PET imaging ligands for Galectin-3, which is a protein overexpressed in pancreatic cancer tumors. We uncover several structural features for the ligands that can be used to improve their binding and efficacy. Second, we discuss the AlkB family of enzymes. AlkB is the E. coli DNA repair protein for alkylation damage, and has human homologues with slightly different functions and substrates. Each has a conserved active site with a catalytic iron and a coordinating His...His...Asp triad. We have applied molecular dynamics (MD) to investigate the effect of a novel single nucleotide polymorphism for AlkBH7, which is correlated with prostate cancer and has an unknown function. We show that the mutation leads to active site distortion, which has been confirmed by experiments. Thirdly, we investigate the unfolding of hen egg white lysozyme in 90% ethanol solution and low pH, to show the initial steps of unfolding from a native-like state to the disease-associated beta-sheet structure. We compare to mass spectrometry experiments and also show differing pathways based on protonation state. Finally, we discuss three different DNA polymerase systems. DNA polymerases are the primary proteins that replicate DNA during cell division, and have various extra or specific functions. We look at a proofreading-deficient DNA polymerase III mutant, the effects of solvent on DNA polymerase IV's ability to bypass bulky DNA adducts, and a variety of mutations on DNA polymerase kappa.

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31

Marklund, Erik. "Gas-Phase Protein Structure Under the Computational Microscope : Hydration, Titration, and Temperature." Doctoral thesis, Uppsala universitet, Beräknings- och systembiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-151006.

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Although the native environment of the vast majority of proteins is a complex aqueous solution, like the interior of a cell, many analysis methods for assessing chemical and physical properties of biomolecules require the sample to be aerosolized; that is, transferred to the gas-phase. An important example is electrospray-ionization mass spectrometry, which can provide a wide range of information about e.g. biomolecules. That includes structural features, charged sites, and gas-phase equilibrium constants of reactions. To date much of the microscopic detail about the aerosolization process remains beyond the limits of experimental observation. How is the gas-phase structure of a protein related to the solution-phase structure? How transferable are observations done in the gas phase to solution? On the basis of classical molecular-dynamics simulations this thesis reveals important features of gas-phase biomolecular structure near the end of the the aerosolization process, the relation between gas-phase structure and native structure, microscopic detail about the de-wetting of gas-phase biomolecules, and the impact of temperature and residual solvent on structure preservation. Residual solvent on proteins is shown to have a stabilizing effect on proteins, in part because it allows the scarcely hydrated protein to cool through solvent evaporation, but also because part of the solvent provides structural support by hydrogen bonding to the protein. The gas-phase structure of micellar aggregates is seen to depend on composition, where some types of lipids cause rapid micelle inversion, whereas others maintain much of their collective structure when transferred to the gas phase. The thesis also addresses proton-transfer reactions, which have an impact on the biophysical aspects of proteins, both in the gas phase and in solution. The thesis presents a computationally efficient method for including proton-transfer reactions in classical molecular-dynamics simulations, which expands the range of scientific problems that can be addressed with molecular dynamics.

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32

Schwing, Gregory John. "Implementation of Replica Exchange with Dynamic Scaling in GROMACS 2018." ScholarWorks@UNO, 2018. https://scholarworks.uno.edu/honors_theses/117.

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This is a problem of sampling. The number of classical states of an N-body system grows with O( 3 ^ N ). To sample this space, advanced techniques are required. Replica Exchange (RE), also known as parallel tempering, is an example that uses parallelization, and Hamiltonian Replica Exchange is a subset of RE that scales the energy of the replicas. The number of simulations required grows at O( N^(1/2) ), where N is number of atoms in the system. Replica Exchange with Dynamical Scaling (REDS) attempts to address this problem to decrease computational cost. It has been shown to increase efficiency 10-fold. We implemented REDS in GROMACS 2018. (Abraham 2015) All changes to the source code were written in the form of parallel methods. Scripts were written in Python and Perl to automate the experiment entirely. An exchange connects a region of high energy space, far above the surface of the landscape, to low energy space, which approaches the surface of the landscape, which represents the natural conformational progression of the molecule. Using REDS we were able to achieve exchanges at temperatures spaced too far apart to exchange using normal RE. Ergo, the flexibility of dynamical scaling allowed regions of phase space that would have gone unsampled to be mapped, addressing our initial problem of sampling.

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33

Dabdoub, Shareef Majed. "Applied Visual Analytics in Molecular, Cellular, and Microbiology." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1322602183.

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34

Dinescu, Adriana. "Modeling wild type and mutant glutathione synthetase." Thesis, University of North Texas, 2004. https://digital.library.unt.edu/ark:/67531/metadc5556/.

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Glutathione syntethase (GS) is an enzyme that belongs to the ATP-grasp superfamily and catalyzes the second step in the biosynthesis of glutathione. GS has been purified and sequenced from a variety of biological sources; still, its exact mechanism is not fully understood. Four highly conserved residues were identified in the binding site of human GS. Additionally, the G-loop residues that close the active site during catalysis were found to be conserved. Since these residues are important for catalysis, their function was studied computationally by site-directed mutagenesis. Starting from the reported crystal structure of human GS, different conformations for the wild type and mutants were obtained using molecular dynamics technique. The key interactions between residues and ligands were detected and found to be essential for enzyme activity.

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35

Stelzl, Lukas Sebastian. "Studying marcomolecular transitions by NMR and computer simulations." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:6e4bbe06-fc58-471b-a932-d940fe78b9a5.

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Macromolecular transitions such as conformational changes and protein-protein association underlie many biological processes. Conformational changes in the N-terminal domain of the transmembrane protein DsbD (nDsbD) were studied by NMR and molecular dynamics (MD) simulations. nDsbD supplies reductant to biosynthetic pathways in the oxidising periplasm of Gram-negative bacteria after receiving reductant from the C-terminal domain of DsbD (cDsbD). Reductant transfer in the DsbD pathway happens via protein-protein association and subsequent thiol-disulphide exchange reactions. The cap loop shields the active-site cysteines in nDsbD from non-cognate oxidation, but needs to open when nDsbD bind its interaction partners. The loop was rigid in MD simulations of reduced nDsbD. More complicated dynamics were observed for oxidised nDsbD, as the disulphide bond introduces frustration which led to loop opening in some trajectories. The simulations of oxidised and reduced nDsbD agreed well with previous NMR spin-relaxation and residual dipolar coupling measurements as well as chemical shift-based torsion angle predictions. NMR relaxation dispersion experiments revealed that the cap loop of oxidised nDsbD exchanges between a major and a minor conformation. The differences in their conformational dynamics may explain why oxidised nDsbD binds its physiological partner cDsbD much tighter than reduced nDsbD. The redox-state dependent interaction between cDsbD and nDsbD is thought to enhance turnover. NMR relaxation dispersion experiments gave insight into the kinetics of the redox-state dependent interaction. MD simulations identified dynamic encounter complexes in the association of nDsbD with cDsbD. The mechanism of the conformational changes in the transport cycle of LacY were also investigated. LacY switches between periplasmic open and cytoplasmic open conformations to transport sugars across the cell membrane. Two mechanisms have been proposed for the conformational change, a rocker-switch mechanism based on rigid body motions and an “airlock” like mechanism in which the transporter would switch conformation via a fully occluded structure. In MD simulations using the novel dynamics importance sampling approach such a fully occluded structure was found. The simulations argued against a strict “rocker-switch” mechanism.

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36

Harrison, Ryan M. "Molecular biophysics of strong DNA bending and the RecQ DNA helicase." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:f02fc167-b705-4275-a413-21d13b5d94c3.

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Molecular biophysics is a rapidly evolving field aimed at the physics-based investigation of the biomolecular processes that enable life. In this thesis, we explore two such processes: the thermodynamics of DNA bending, and the mechanism of the RecQ DNA helicase. A computational approach using a coarse-grained model of DNA is employed for the former; an experimental approach relying heavily on single-molecule fluorescence for the latter. There is much interest in understanding the physics of DNA bending, due to both its biological role in genome regulation and its relevance to nanotechnology. Small DNA bending fluctuations are well described by existing models; however, there is less consensus on what happens at larger bending fluctuations. A coarse-grained simulation is used to fully characterize the thermodynamics and mechanics of duplex DNA bending. We then use this newfound insight to harmonize experimental results between four distinct experimental systems: a 'molecular vise', DNA cyclization, DNA minicircles and a 'strained duplex'. We find that a specific structural defect present at large bending fluctuations, a 'kink', is responsible for the deviation from existing theory at lengths below about 80 base pairs. The RecQ DNA helicase is also of much biological and clinical interest, owing to its essential role in genome integrity via replication, recombination and repair. In humans, heritable defects in the RecQ helicases manifest clinically as premature aging and a greatly elevated cancer risk, in disorders such as Werner and Bloom syndromes. Unfortunately, the mechanism by which the RecQ helicase processes DNA remains poorly understood. Although several models have been proposed to describe the mechanics of helicases based on biochemical and structural data, ensemble experiments have been unable to address some of the more nuanced questions of helicase function. We prepare novel substrates to probe the mechanism of the RecQ helicase via single-molecule fluorescence, exploring DNA binding, translocation and unwinding. Using this insight, we propose a model for RecQ helicase activity.

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37

Nobrega, Robert P. "Early Folding Biases in the Folding Free-Energy Surface of βα-Repeat Proteins: A Dissertation". eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/723.

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Early events in folding can determine if a protein is going to fold, misfold, or aggregate. Understanding these deterministic events is paramount for de novo protein engineering, the enhancement of biopharmaceutical stabilities, and understanding neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimer's disease. However, the physicochemical and structural biases within high energy states of protein biopolymers are poorly understood. A combined experimental and computational study was conducted on the small β/α-repeat protein CheY to determine the structural basis of its submillisecond misfolding reaction to an off-pathway intermediate. Using permutations, we were able to discriminate between the roles of two proposed mechanisms of folding; a nucleation condensation model, and a hydrophobic collapse model driven by the formation of clusters of isoleucine, leucine, and valine (ILV) residues. We found that by altering the ILV cluster connectivity we could bias the early folding events to either favor on or off-pathway intermediates. Structural biases were also experimentally observed in the unfolded state of a de novo designed synthetic β/α-repeat protein, Di-III_14. Although thermodynamically and kinetically 2-state, Di-III_14 has a well structured unfolded state that is only observable under native-favoring conditions. This unfolded state appears to retain native-like structure, consisting of a hydrophobic 7 core (69% ILV) stabilized by solvent exposed polar groups and long range electrostatic interactions. Together, these results suggest that early folding events are largely deterministic in these two systems. Generally, low contact order ILV clusters favor local compaction and, in specific cases, long range electrostatic interactions may have stabilizing effects in higher energy states.

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38

Nobrega, Robert P. "Early Folding Biases in the Folding Free-Energy Surface of βα-Repeat Proteins: A Dissertation". eScholarship@UMMS, 2007. http://escholarship.umassmed.edu/gsbs_diss/723.

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Early events in folding can determine if a protein is going to fold, misfold, or aggregate. Understanding these deterministic events is paramount for de novo protein engineering, the enhancement of biopharmaceutical stabilities, and understanding neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimer's disease. However, the physicochemical and structural biases within high energy states of protein biopolymers are poorly understood. A combined experimental and computational study was conducted on the small β/α-repeat protein CheY to determine the structural basis of its submillisecond misfolding reaction to an off-pathway intermediate. Using permutations, we were able to discriminate between the roles of two proposed mechanisms of folding; a nucleation condensation model, and a hydrophobic collapse model driven by the formation of clusters of isoleucine, leucine, and valine (ILV) residues. We found that by altering the ILV cluster connectivity we could bias the early folding events to either favor on or off-pathway intermediates. Structural biases were also experimentally observed in the unfolded state of a de novo designed synthetic β/α-repeat protein, Di-III_14. Although thermodynamically and kinetically 2-state, Di-III_14 has a well structured unfolded state that is only observable under native-favoring conditions. This unfolded state appears to retain native-like structure, consisting of a hydrophobic 7 core (69% ILV) stabilized by solvent exposed polar groups and long range electrostatic interactions. Together, these results suggest that early folding events are largely deterministic in these two systems. Generally, low contact order ILV clusters favor local compaction and, in specific cases, long range electrostatic interactions may have stabilizing effects in higher energy states.

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39

Viveca, Lindahl. "Optimizing sampling of important events in complex biomolecular systems." Doctoral thesis, KTH, Fysik, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-217837.

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Proteins and DNA are large, complex molecules that carry out biological functions essential to all life. Their successful operation relies on adopting specific structures, stabilized by intra-molecular interactions between atoms. The spatial and temporal resolution required to study the mechanics of these molecules in full detail can only be obtained using computer simulations of molecular models. In a molecular dynamics simulation, a trajectory of the system is generated, which allows mapping out the states and dynamics of the molecule. However, the time and length scales characteristic of biological events are many orders of magnitude larger than the resolution needed to accurately describe the microscopic processes of the atoms. To overcome this problem, sampling methods have been developed that enhance the occurrence of rare but important events, which improves the statistics of simulation data. This thesis summarizes my work on developing the AWH method, an algorithm that adaptively optimizes sampling toward a target function and simultaneously finds and assigns probabilities to states of the simulated system. I have adapted AWH for use in molecular dynamics simulations. In doing so, I investigated the convergence of the method as a function of its input parameters and improved the robustness of the method. I have also worked on a generally applicable approach for calculating the target function in an automatic and non-arbitrary way. Traditionally, the target is set in an ad hoc way, while now sampling can be improved by 50% or more without extra effort. I have also used AWH to improve sampling in two biologically relevant applications. In one paper, we study the opening of a DNA base pair, which due to the stability of the DNA double helix only very rarely occurs spontaneously. We show that the probability of opening depends on both nearest-neighbor and longer-range sequence effect and furthermore structurally characterize the open states. In the second application the permeability and ammonia selectivity of the membrane protein aquaporin is investigated and we show that these functions are sensitive to specific mutations.

QC 20171117

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40

Trovato, Fabio. "Molecular Dynamics Simulations of biopolymers within the cell environment: Minimalist models for the Nucleic Acids and Green Fluorescent Proteins in the cytoplasm." Doctoral thesis, Scuola Normale Superiore, 2013. http://hdl.handle.net/11384/85896.

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From the introduction: "... This thesis reports on simplified models (`coarse grained') of proteins and nucleic acids in which many degrees of freedom (DOF) are eliminated, to speed up the computation of the interactions. The resolution of these models is minimal, with respect to the representation of the phenomena studied, in order to extend the time and length scales accessible to atomistic simulations. Furthermore the existence of a coarse- to fine-grain backmapping ensures to switch between diff erent resolutions of the same system, naturally leading to an integrative approach used for multi-scale simulations. Careful parameterization and optimization of the interactions between the interactive centers additionally allow to overcome the limitations of the reductionist first stage by including directly experimental and theoretical data.

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41

Sánchez, Martínez Melchor. "Protein Flexibility: From local to global motions. A computational study." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/288044.

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Proteins are flexible entities, and thus move. Its function is closely related to the flexibility. To carry out any function is necessary a conformational change. As protein motions imply an exchange of conformations, protein dynamics is also known as Protein Conformational Dynamics. The fluctuations between the different proteic configurations can be classified according to the length-scale, the time-scale and the amplitude and directionality of them. The movement could be local movement, involving only the rearrangement of a few amino- acid side chains or even backbone, or it may be a large, global movement, modulating the allostery or the conformational transitions, and even involve folding of the entire protein. Generally local motions are also fast and small amplitude motions whereas global motions are associated with slow and large amplitude motions. All these motions encompassed into protein dynamics are governed by the features of the underlying energy landscape. To fully describe a protein, a multidimensional and rugged energy landscape defining the relative probabilities of the conformational states (thermodynamics) and the energy barriers between them (kinetics) is required. To understand proteins in action, the fourth dimension, time, must be added. As these motions are important for the proteic function a deep understanding is required. To do that in this thesis we have tried to answer several questions related to these dynamical phenomena. Concretely we have performed local studies, related to enzyme catalysis and protein damage, and globally, with the determination and analysis of protein conformational ensembles. These studies have been developed using methods of computational chemistry, computational biochemistry and computational biophysics, which have proven to be very useful tools when studying the protein dynamics. Computational methods are an efficient and useful tool to characterize protein motions. However the current computational approaches present limitations and to solve them the incorporation of experimental data and its correct interpretation is crucial. This necessity of be complemented comes from different sides: 1) from experiments to computations and 2) from computations to experiments. The convergence of experimental and computational techniques to the same point is key to achieve a deep understanding of protein dynamics.
La presente tesis se centra en el estudio computacional de la dinámica de las proteínas. Las proteínas son entidades flexibles y como tales se mueven. Este movimiento es indispensable y esta directamente relacionado con su función. La dinámica de las proteínas se puede dividir en dos grandes bloques conceptuales según el número de átomos involucrados, la escala de tiempo en que tiene lugar y la amplitud y dirección de la misma. Debido a la importancia de estos fenómenos, emerge la necesidad de tener un conocimiento profundo sobre los mismos. Debido a ello, en esta tesis doctoral se ha tratado de dar respuesta a varios fenómenos observados en relación directa con la dinámica de las proteínas. Concretamente, hemos realizado estudios a nivel local, de 'centro activo', relacionados con la catálisis enzimática y el daño proteico, así como a nivel global, con la determinación y el análisis de conjuntos conformacionales de proteínas. Estos estudios, se han desarrollado usando métodos propios de la química, la bioquímica y la biofísica computacionales, los cuales se han mostrado como herramientas muy útiles a la hora de estudiar la dinámica. De todos ellos, de forma general, podemos concluir que los métodos computacionales son una herramienta eficaz y util para caracterizar la dinámica de las proteínas. Sin embargo, los métodos computacionales actuales presentan limitaciones y para resolverlos la incorporación de datos experimentales as como su correcta interpretación es crucial. Pero aunque los metodos computacionales necesitan de los experimentales, esta necesidad también se da de manera opuesta. La convergencia de los métodos experimentales y computacionales es clave para poder profundizar en el conocimiento de la dinámica de las proteínas.

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42

Sahai, Michelle Asha. "Computational studies of ligand-water mediated interactions in ionotropic glutamate receptors." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:b86d2f5a-3554-44c0-b985-5693241369ec.

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Careful treatment of water molecules in ligand-protein interactions is required in many cases if the correct binding pose is to be identified for molecular docking. Water can form complex bridging networks and can play a critical role in dictating the binding mode of ligands. A particularly striking example of this can be found in the ionotropic glutamate receptors (iGluRs), a family of ligand gated ion channels that are responsible for a majority of the fast synaptic neurotransmission in the central nervous system that are thought to be essential in memory and learning. Thus, pharmacological intervention at these neuronal receptors is a valuable therapeutic strategy. This thesis relies on various computational studies and X-ray crystallography to investigate the role of ligand-water mediated interactions in iGluRs bound to glutamate and α-amino-3-hydroxy-5-methyl-4- isoxazole-propionic acid (AMPA). Comparative molecular dynamics (MD) simulations of each subtype of iGluRs bound to glutamate revealed that crystal water positions were reproduced and that all but one water molecule, W5, in the binding site can be rearranged or replaced with water molecules from the bulk. Further density functional theory calculations (DFT) have been used to confirm the MD results and characterize the energetics of W5 and another water molecule implicated in influencing the dynamics of a proposed switch in these receptors. Additional comparative studies on the AMPA subtypes of iGluRs show that each step of the calculation must be considered carefully if the results are to be meaningful. Crystal structures of two ligands, glutamate and AMPA revealed two distinct modes of binding when bound to an AMPA subtype of iGluRs, GluA2. The difference is related to the position of water molecules within the binding pocket. DFT calculations investigated the interaction energies and polarisation effects resulting in a prediction of the correct binding mode for glutamate. For AMPA alternative modes of binding have similar interaction energies as a result of a higher internal energy than glutamate. A combined MD and X-ray crystallographic study investigated the binding of the ligand AMPA in the AMPA receptor subtypes. Analysis of the binding pocket show that AMPA is not preserved in the crystal bound mode and can instead adopt an alternative mode of binding. This involves a displacement of a key water molecule followed by AMPA adopting the pose seen by glutamate. Thus, this thesis makes use of various studies to assess the energetics and dynamics of water molecules in iGluRs. The resulting data provides additional information on the importance of water molecules in mediating ligand interactions as well as identifying key water molecules that can be useful in the de novo design of new selective drugs against iGluRs.

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43

Ihms, Elihu Carl. "Integrative Investigation and Modeling of Macromolecular Complexes." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1429547886.

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44

Rzepala, Wojciech. "Interactions of carbon nanotubes and lipid bilayers." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:58cd5321-c61c-4594-b74d-8ca6f507c48f.

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The biological membrane, which is composed of a lipid bilayer embedded with numerous proteins, defines the cell boundary, separating the cell interior from the external environment. It serves as a gatekeeper and entry point for various molecular and ionic species. This thesis describes experimental and simulation studies of the interactions of carbon nanotubes (CNTs) with model membranes (lipid bilayers). The unique properties of CNTs make them ideal candidates for many nanotechnological applications. They can, however, pose a potential risk as toxins. While research into the positive benefits of CNTs continues, very little is known about their basic interactions with cellular components. It is particularly important to understand the interaction of CNTs with biological membranes, which form the primary physical barrier surrounding a cell. Coarse grained molecular dynamics (MD) simulations and atomic force microscopy (AFM) have been used to study the interactions of CNTs and lipid bilayers. They are investigated in a controlled manner using MD simulations, while AFM has allowed the controlled approach-to-contact and insertion of CNTs into bilayers. A number of effects are reported, including lipid creep along the CNT and bilayer thickening upon contact. The robustness of this response is established using different force fields and lipid species. The experimental results show an unusual reaction to mechanical indentation, and are further backed by MD simulations. The lipid bilayer response to multiple CNTs is studied and the effects of CNTs on bilayer conformation and lipid diffusion are reported. CNT internalisation from the solvent is observed in the simulations. Indeed, many of the observed phenomena are reminiscent of those known from the field of membrane protein. This project focuses on understanding the basic molecular interactions of CNTs with lipid bilayers and addresses the gap between experimental and computational work.

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45

Tengel, Tobias. "Studies of protein structure, dynamics and protein-ligand interactions using NMR spectroscopy." Doctoral thesis, Umeå : Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1472.

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46

Gonzalez, Walter G. "Protein-Ligand Interactions and Allosteric Regulation of Activity in DREAM Protein." FIU Digital Commons, 2016. http://digitalcommons.fiu.edu/etd/2503.

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Downstream regulatory antagonist modulator (DREAM) is a calcium sensing protein that co-assembles with KV4 potassium channels to regulate ion currents as well as with DNA in the nucleus, where it regulates gene expression. The interaction of DREAM with A-type KV4 channels and DNA has been shown to regulate neuronal signaling, pain sensing, and memory retention. The role of DREAM in modulation of pain, onset of Alzheimer’s disease, and cardiac pacemaking has set this protein as a novel therapeutic target. Moreover, previous results have shown a Ca2+ dependent interaction between DREAM and KV4/DNA involving surface contacts at the N-terminus of DREAM. However, the mechanisms by which Ca2+ binding at the C-terminus of DREAM induces structural changes at the C- and N-terminus remain unknown. Here, we present the use of biophysics and biochemistry techniques in order to map the interactions of DREAM and numerous small synthetic ligands as well as KV channels. We further demonstrate that a highly conserved network of aromatic residues spanning the C- and N-terminus domains control protein dynamics and the pathways of signal transduction on DREAM. Using molecular dynamics simulations, site directed mutagenesis, and fluorescence spectroscopy we provide strong evidence in support of a highly dynamic mechanism of signal transduction and regulation. A set of aromatic amino acids including Trp169, Phe171, Tyr174, Phe218, Phe235, Phe219, and Phe252 are identified to form a dynamic network involved in propagation of Ca2+ induced structural changes. These amino acids form a hydrophobic network connecting the N- and C-terminus domains of DREAM and are well conserved in other neuronal calcium sensors. In addition, we show evidence in support of a mechanism in which Ca2+ signals are propagated towards the N-terminus and ultimately lead to the rearrangement of the inactive EF-hand 1. The observed structural motions provide a novel mechanism involved in control of the calcium dependent KV4 and DNA binding. Altogether, we provide the first mechanism of intramolecular and intermolecular signal transduction in a Ca2+ binding protein of the neuronal calcium sensor family.

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47

Mahajan, Rahul. "Gβγ acts at an inter-subunit cleft to activate GIRK1 channels". VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/3307.

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Heterotrimeric guanine nucleotide-binding proteins (G-proteins) consist of an alpha subunit (Gα) and the dimeric beta-gamma subunit (Gβγ). The first example of direct cell signaling by Gβγ was the discovery of its role in activating G-protein regulated inwardly rectifying K+ (GIRK) channels which underlie the acetylcholine-induced K+ current responsible for vagal inhibition of heart rate. Published crystal structures have provided important insights into the structures of the G-protein subunits and GIRK channels separately, but co-crystals of the channel and Gβγ together remain elusive and no specific reciprocal residue interactions between the two proteins are currently known. Given the absence of direct structural evidence, we attempted to identify these functionally important channel-Gβγ interactions using a computational approach. We developed a multistage computational docking algorithm that combines several known methods in protein-protein docking. Application of the docking protocol to previously published structures of Gβγ and GIRK1 homomeric channels produced a clear signal of a favored binding mode. Analysis of this binding mode suggested a mechanism by which Gβγ promotes the open state of the channel. The channel-Gβγ interactions predicted by the model in silico could be disrupted in vitro by mutation of one protein and rescued by additional mutation of reciprocal residues in the other protein. These interactions were found to extend to agonist induced activation of the channels as well as to activation of the native heteromeric channels. Currently, the structural mechanism by which Gβγ regulates the functional conformations of GIRK channels or of any of its membrane-associated effector proteins is not known. This work shows the first evidence for specific reciprocal interactions between Gβγ and a GIRK channel and places these interactions in the context of a general model of intracellular regulation of GIRK gating.

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48

Migliore, Mattia. "Recherche par modélisaion moléculaire de signatures RMN et DC caractéristiques pour les coudes β et y dans les peptides bioactifs. Characterization of β-turns by electronic circular dichroism spectroscopy : a coupled molecular dynamics and time-dependent density functional theory computational study". Thesis, Normandie, 2020. http://www.theses.fr/2020NORMR001.

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Ce travail de thèse a porté sur la recherche, par modélisation moléculaire, de signatures RMN et DC caractéristiques pour les coudes γ et β dans les peptides bioactifs. Les coudes γ et β, avec les hélices, constituent des motifs privilégiés de reconnaissance des peptides bioactifs par leurs cibles. Or, bien qu’il existe, au sein des structures de polypeptides, plusieurs catégories de coudes possédant des géométries différentes (2 types de coudes γ et 12 types de coudes β), peu d’outils expérimentaux sont disponibles pour aider à leur distinction. Ainsi, seuls 4 types de coudes β (I, I’, II et II’) ont, pour l’instant été caractérisés par RMN et il n’existe pas de spectre DC de référence fiable pour aucun motif de type coude. Dans un premier temps, afin d’élargir la base de données de RMN pour tous les types de coudes β (I, I’, II, II’, IV₁, IV₂, IV₃, IV₄, Via1, Via2, VIb et VIII) et γ (classique et inverse), nous avons analysé les paramètres structuraux caractéristiques de RMN (distances inter-hydrogènes et constantes de couplages ᶾJʜɴ-ʜꭤ) sur un ensemble de peptides modèles, extraits de la banque de protéines PDB et représentatifs de ces coudes. Les analyses des distances inter-hydrogènes ont permis d’identifier des signatures RMN caractéristiques pour différencier les deux types de coudes γ et certains types de coudes β (IV₁, IV₂,, VIb et VIII). La constante de couplage ᶾJʜɴ-ʜꭤ pourra servir à confirmer l’identification et à lever des ambiguïtés. Dans un second temps, en couplant dynamique moléculaire et TDDFT, nous avons simulé les spectres de DC de référence de peptides modèles adoptant des conformations de coudes β de type I, I’, II et II’. Les simulations ont permis de discerner deux familles de spectres DC caractéristiques : les types I/II’ d’un côté et les types I’/II de l’autre. L’ensemble de ces résultats indique que les coudes ne présentent pas nécessairement les mêmes signatures pour les deux techniques. La combinaison des signatures discriminantes de RMN et de DC pourrait donc permettre une meilleure identification des natures et des différents types de coudes
The aim of this work is to identify NMR and CD characteristic patterns for β- and γ-turns in bioactive peptides by molecular modelling. With helices, β- and γ-turns constitute favoured recognition motifs in bioactive peptides by their targets. Even though several classes of turns with different geometries exist in polypeptide structures (2 γ-turn types and 12 β-turn types), few experimental tools are available for their characterization. Thus, only 4 types of β-turns (I, I’, II et II’) have been, at present, described by NMR and there are no reliable reference CD spectra for turns. In order to extend the NMR data for all β- and γ-turn types, we analyzed NMR structural parameters (inter-hydrogen distances and ᶾJʜɴ-ʜꭤ coupling constants) in a representative peptide model dataset extracted from the PDB. The inter-hydrogen distance analysis allowed to identify specific NMR patterns for the two γ-turn types and for four β-turn types (IV₁, IV₂,, VIb and VIII). ᶾJʜɴ-ʜꭤ coupling constant may be used to confirm the identification and to remove ambiguities. Then, we simulated the reference CD spectra of model peptides adopting type I, I’, II and II’ β-turn conformations by combining molecular dynamic simulations and TDDFT computations. These computations allowed to determine two families of specific CD spectra : types I/II’, on one side and types I’/II, on the other. All these results indicate that the turns do not present the same patterns in both techniques. The combination of NMR and CD could therefore allow a better identification of the nature and the different types of turns

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49

Fu, Josephine K. Y. "Functional characterization of the teleost multiple tissue (tmt) opsin family and their role in light detection." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:39bc18bb-16cb-4549-94cd-5f872daafe7e.

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In addition to a central circadian clock in the suprachiasmatic nucleus (SCN), zebrafish (Danio rerio) have local clock systems in their peripheral tissues. These peripheral tissues express a complement of clock genes that can be synchronized with the 24 h light/dark cycle and thus may be entrained by light. To date, teleost multiple tissue (tmt) opsin identified from Fugu rubripes and Danio rerio is the only opsin that has been proposed as a candidate to mediate this cellular photoentrainment (Moutsaki et al., 2003). Here we report the discovery of a multigene family of tmt opsins found not only in the teleost fishes, but in vertebrates,including amphibians, birds, reptiles, and some mammals. Phylogenetic analysis demonstrated that this gene family consists of three main classes, tmtI, tmtII and tmtIII, with each duplicating further to give two paralogues in the zebrafish genome. Their predicted amino acid sequences contain most of the characteristic features for the function of a photopigment opsin, as well as seven transmembrane segments indicative of a G protein coupled receptor (GPCR) superfamily. Significantly, reverse transcription polymerase chain reaction (RT-PCR) reveals that the tmt opsin genes in zebrafish are both temporally and spatially regulated. To investigate if these tmt photopigments mediate light-activated currents in cells, each opsin was expressed in vitro and the responses characterised by calcium imaging, whole-cell patch clamp electrophysiology, UV-Vis spectrophotometric analysis, and bioluminescence reporter assay. Collectively, these data suggest that some of the opsin photoproteins signal via Gi-type G protein pathway. Interestingly, the spectral analysis obtained shows that most tmt opsins tested are UV-sensitive when reconstituted in vitro with 11-cis and all-trans retinal, indicating an intrinsic bistable dynamics. Using site directed mutagenesis on one of the tmt opsins, tmt10, the potential spectral tuning sites involved in UV detection were tested. As part of this study, tmt opsin cDNAs were isolated from three populations of Mexican tetra (Astyanax mexicanus): surface, Pachon and Steinhardt. This allowed for a direct comparison between the tmt opsins present in the dark adapted species (cavefish) versus those of the light adapted species (zebrafish). It is hoped that the findings from this project will contribute to our understanding of non-visual light detection in fish and the evolution of their non-image forming photoreception.

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Ragland, Debra A. "The Structural Basis for the Interdependence of Drug Resistance in the HIV-1 Protease." eScholarship@UMMS, 2012. http://escholarship.umassmed.edu/gsbs_diss/879.

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The human immunodeficiency virus type 1 (HIV-1) protease (PR) is a critical drug target as it is responsible for virion maturation. Mutations within the active site (1°) of the PR directly interfere with inhibitor binding while mutations distal to the active site (2°) to restore enzymatic fitness. Increasing mutation number is not directly proportional to the severity of resistance, suggesting that resistance is not simply additive but that it is interdependent. The interdependency of both primary and secondary mutations to drive protease inhibitor (PI) resistance is grossly understudied. To structurally and dynamically characterize the direct role of secondary mutations in drug resistance, I selected a panel of single-site mutant protease crystal structures complexed with the PI darunavir (DRV). From these studies, I developed a network hypothesis that explains how mutations outside the active site are able to perpetuate changes to the active site of the protease to disrupt inhibitor binding. I then expanded the panel to include highly mutated multi-drug resistant variants. To elucidate the interdependency between primary and secondary mutations I used statistical and machine-learning techniques to determine which specific mutations underlie the perturbations of key inter-molecular interactions. From these studies, I have determined that mutations distal to the active site are able to perturb the global PR hydrogen bonding patterns, while primary and secondary mutations cooperatively perturb hydrophobic contacts between the PR and DRV. Discerning and exploiting the mechanisms that underlie drug resistance in viral targets could proactively ameliorate both current treatment and inhibitor design for HIV-1 targets.

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Дисертації з теми "Proteins Molecular Dynamics Computational Biophysics"

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