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Title: SIMULATION OF H2A.B CONTAINING HISTONE VARIANT NUCLEOSOME
Creator: Kohestani, Havva
Date: 2019
Description: The H2A.B histone is a highly evolving vertebrate specific variant of the H2A histone family. It has been implicated in increased gene...
Show moreThe H2A.B histone is a highly evolving vertebrate specific variant of the H2A histone family. It has been implicated in increased gene expression, and experiments have shown that incorporation of H2A.B into nucleosomes results in more extended structures with fewer wrapped DNA base pairs. To study the molecular mechanisms of H2A.B, we have performed a series of conventional and enhanced sampling molecular dynamics simulation of H2A.B and canonical H2A containing nucleosomes.Results of adaptively biased molecular simulations show that substitution of canonical H2A with H2A.B results in geometrical changes such as unwrapping of 10 to 15 base pairs of DNA on each side of the nucleosome and an increase in the diameter and radius of gyration, which is in agreement with previous AFM, FRET, and SAXS experiments. DNA unwinding is energetically favorable in H2A.B containing compared to canonical nucleosomes, while in both systems we observe a wide range of sampling over various structures of DNA. H3 histone tails excluded simulations, show the importance and effect of N-terminal residues of H3 histones on attachment of DNA at the entry/exit sites to nucleosome protein core. Clustering and hydrogen bond analysis suggest that introduction of H2A.B to nucleosome systems triggers mechanisms leading to rearrangement of hydrogen bond network which may influence the pattern and intensity of interactions between DNA-protein and protein-protein complexes.
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Title: Toward a Comprehensive Atomistic View of Bacterial Outer Membrane Remodeling and Antimicrobial Peptide Susceptibility
Creator: Rice, Amy
Date: 2019
Description: The cell membrane is arguably one of the most important and complex features of a cell, serving to demarcate “self” from “environment” and...
Show moreThe cell membrane is arguably one of the most important and complex features of a cell, serving to demarcate “self” from “environment” and selectively controlling the flow of material in and out of the cell. Bacterial cell membranes are of additional interest since they serve as the first point of contact for antibiotic drugs or other antimicrobial agents. In this work, I use atomistic molecular dynamics simulations to study factors that alter bacterial susceptibility to antimicrobial agents and their environment, with the goal of better understanding how bacteria are able to modulate their sensitivity.First, I present results from a series of simulations of antimicrobial peptides (AMP) interacting with phospholipid bilayers to elucidate the observed interaction differences between arginine and lysine-containing AMPs. Simulations show that the extensive interaction with arginine is due in part to arginine's strong atomic charge distribution, rather than being purely an effect of the greater hydrogen bond capacity. These results highlight the role of charge and hydrogen bond strength in peptide bilayer insertion, and offer potential insights for designing more potent analogues in the future.Next, the effects of bacterial lipopolysaccharide (LPS) modifications are examined, first to understand study how three key modifications observed in S. enterica affect bilayer properties, as well as to examine the role these modifications play in AMP resistance. We hypothesize that defects at the crystalline/liquid-ordered boundary facilitate LL-37 intercalation into the outer membrane, whereas LPS modification protects against this process by having already increased crystallinity and packing efficiency. These results further improve our understanding of outer membrane chemical properties and help elucidate how outer membrane modification systems are able to alter bacterial virulence and susceptibility. Lastly, I investigate the effects of ion type and phosphate charge on four distinct LPS types. Simulations show that bilayer properties are highly influenced by the choice of cation type, ion parameterization, and phosphate group charges. Additional free energy perturbation simulations predict that the protonated LPS state should dominate at physiological pH, in contrast to the deprotonated state utilized by many LPS force fields. Overall, these results reveal inaccuracies in the existing LPS force fields and establish guidelines to better reproduce experimental LPS membrane properties.
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Title: Computational study on the heme scavenging ability of Staphylococcus aureus IsdH receptor: Utilizing molecular dynamics to understand an unknown mechanism
Creator: Clayton, Joseph Alan
Date: 2021
Description: Methicillin-resistant Staphylococcus aureus (MRSA) has become an infamous pathogen with infection rates that have declined slowly in recent...
Show moreMethicillin-resistant Staphylococcus aureus (MRSA) has become an infamous pathogen with infection rates that have declined slowly in recent years. S. aureus requires iron as a metabolic nutrient during infection and obtains this nutrient through an iron-regulated surface-determinant (Isd) system that extracts iron from the host’s heme stored in hemoglobin (Hb) through near iron transporter (NEAT) domains. This work concentrates on studying the second and third NEAT domains of IsdH by utilizing atomistic molecular dynamics to probe the heme scavenging process; in collaboration with the Clubb Group at UCLA, we discover key functional regions of IsdH and describe fundamental interdomain dynamics. In addition, I investigate a conventional computational method to describe protein dynamics and propose an alternative that aims to alleviate computational effort by incorporating experimental data.
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Title: Kinetic and Structural Characterization of the Vibrio cholerae Flavin Transferase ApbE
Creator: Fang, Xuan
Date: 2019
Description: Cholera has long been a global concern and in the past decades traditional antibiotic treatments have failed due to the emergence of the...
Show moreCholera has long been a global concern and in the past decades traditional antibiotic treatments have failed due to the emergence of the antibiotic-resistance of its causative agent, V. cholerae. The resistance is mainly supported by a transmembrane electrochemical gradient of Na+ produced by the respiratory complex Na+-NQR coupled with an internal electron transfer pathway. The assembly and function of Na+-NQR is fulfilled by ApbE, the only known flavin transferase which covalently attaches two FMN molecules to the complex as part of its electron transport chain. Hence, ApbE is closely associated with the cause of antibiotic resistance. Because it does not have any human homologues, ApbE becomes an excellent drug target. In this work, we have investigated the physical properties of the enzyme and clarified its substrate specificity and pH dependence. For instance, our experiments indicate that divalent cations are essential for ApbE function, and that the selectivity depends largely on the size and the coordination sphere of the cation. Our data also show that ApbE regulation by pH, ADP and potassium is an important mechanism that enhances the adaptation, survival and colonization of V. cholerae in the small intestine. Moreover, pH dependence, mutagenesis, and steady-state kinetic studies have led us to identify the conserved His257 as a residue with dual roles: substrate binding and catalysis. Furthermore, bi-substrate kinetic studies have also revealed that ApbE follows a random Bi Bi mechanism. Together with structural studies, we propose a reaction mechanism where His257 functions as a base, shedding light into the understanding of the ApbE family.
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Title: Linear Systems Analysis of Molecular Dynamics
Creator: Nicholson, Stanley Anselm
Date: 2023
Description: Most proteins reduce the complexity of atomic motion to stable and coherent structures. Molecular dynamics (MD) has provided swaths of...
Show moreMost proteins reduce the complexity of atomic motion to stable and coherent structures. Molecular dynamics (MD) has provided swaths of trajectory data of proteins. We analyze these trajectories using classical stochastic signal analysis, well established and utilized by engineers. Linear systems analysis operates to uncover linearities given an input and output signal. The coherence function says an input and output are linearly related if and only if the coherence equals one. Analyzing protein motion in the frequency domain allows us to extract a frequency function relating the modes of motion as determined by atomic power spectra. Motivated by biochemistry, we analyze classical interactions like hydrogen bonds and salt bridges and find they act like a linear system, or effective spring. We test our analysis on two protein systems: crambin and the Mu Opioid Receptor (MOR). We extend our results to all pairwise interaction and determine coherent communities of atoms within the MOR. We present various community detection algorithms and demonstrate their validity using common metrics in MD. Identifying rigid and tightly correlated regions of the protein offers great potential in coarse graining protein structure and understanding protein motion.
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Title: AN EXPLORATION INTO THE EFFECTS OF CHROMATIN STRUCTURAL PROTEINS ON THE DYNAMICS AND ENERGETIC LANDSCAPE OF NUCLEOSOME ARCHITECTURES
Creator: Woods, Dustin C
Date: 2022
Description: Comprised of eight core histones wrapped around at least 147 base pairs of DNA, nucleosomes are the fundamental unit the chromatin fiber from...
Show moreComprised of eight core histones wrapped around at least 147 base pairs of DNA, nucleosomes are the fundamental unit the chromatin fiber from which long arrays are built to compact genetic information into the cell nucleus. Structural proteins, such as linker histones (LH) and centromere proteins (CENP), interact with the DNA to dictate the exact architecture of the fiber which can directly influence the regulation of epigentic processes. However, the mechanisms by which structural proteins affect these processes are poorly understood. In this thesis, I will explore the various way in which LHs and CENP-N affect nucleosome and, by extension, chromatin fiber dynamics. First, I present a series of simulations of nucleosomes bound to LHs, otherwise known as chromatosomes, with the globular domain of two LH variants, generic H1 (genGH1) and H1.0 (GH1.0), to determine how their differences influence chromatosome structures, energetics and dynamics. These simulations highlight the thermodynamic basis for different LH binding motifs, and details their physical and chemical effects on chromatosomes. Second, I examine how well the findings above translate from mono-nucleosomes to poly-nucleosome arrays. I present a series of molecular dynamics simulations of octa-nucleosome arrays, based on a cryo-EMstructure of the 30-nm chromatin fiber, with and without the globular domains of the H1 LH to determine how they influence fiber structures and dynamics. These simulations highlight the effects of LH binding on the internal dynamics and global structure of poly- nucleosome arrays, while providing physical insight into a mechanism of chromatin compaction. Third, I took a brief departure from LHs to study the effects that the centromere protein N (CENP-N) has on the poly-nucleosome systems. I present a series of molecular dynamics simulations of CENP-N and di-nucleosome complexes based on cryo- EM and crystal structures provided by Keda Zhou and Karolin Luger. Simulations were conducted with nucleosomes in complex with one, two, and no CENP-Ns. This work, in collaboration with the Karolin Luger Group (University of Colorado – Boulder) and the Aaron Straight Group (Stanford University), represents the first atomistic simulations of this novel complex, providing the foundation for a plethora of future research opportunities exploring centromeric chromatin the effect that its structure and dynamics have on epigenetics. Lastly, I return to the chromatosome to study how DNA sequence affects the free energy surface and detailed mechanism of LH transitions between binding modes. I used umbrella sampling simulations to produce PMFs of chromatosomes wrapped in three different DNA sequences: Widom 601, poly-AT, and poly-CG. This work, my final in the series, represents a culmination of my studies furthering the understanding of biophysical phenomena surrounding LHs and how they can be extrapolated towards epigentic mechanisms. I was able to report on the first PMFs illustrating a previously unknown transition and describe the transition mechanism as it depends on DNA sequence.
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$X-Ray Diffraction Studies of Activation and Relaxation In Fast and Slow Rat Skeletal Muscle$

Title: X-Ray Diffraction Studies of Activation and Relaxation In Fast and Slow Rat Skeletal Muscle
Creator: Gong, Henry M.
Date: 2022
Description: The contractile properties of fast-twitch and slow-twitch skeletal muscles are primarily determined by the myosin isoform content and...
Show moreThe contractile properties of fast-twitch and slow-twitch skeletal muscles are primarily determined by the myosin isoform content and modulated by a variety of sarcomere proteins. X-ray diffraction studies of regulatory mechanisms in muscle contraction have focused predominately on fast- or mixed-fiber muscle with slow muscle being much less studied. Here, we used time-resolved x-ray diffraction to investigate the dynamic behavior of the myofilament proteins in relatively pure slow fiber rat soleus (SOL) and pure fast fiber rat extensor digitorum longus (EDL) muscle during twitch and tetanic contractions at optimal lengths (Lo), 95% Lo, and 90% Lo. Before the delivery of stimulation, reduction in muscle length led to decrease in passive tension. The x-ray reflections upon reduction in length showed no transition in the myosin heads from ordered OFF state, where heads are held close to the thick filament backbone, to disordered ON states, where heads are free to bind to thin filament, in both muscles. When stimulation was delivered to both muscles for twitch contractions at Lo, x-ray signatures indicating the transition of myosin heads to ON states were observed in EDL but not in soleus muscle. During tetanic contractions, changes in the disposition of myosin heads as active tension develops is a cooperative process in EDL muscle whereas in soleus muscle this relationship is less cooperative. Moreover, this high cooperativity was maintained in EDL at all lengths tested here, but cooperativity decreased upon reduction in lengths in soleus. The observed reduced extensibility of the thick filaments in soleus muscles as compared to EDL muscles indicate a molecular basis for this behavior. These data indicate that for the EDL thick filament activation is a cooperative strain-induced mechano-sensing mechanism, whereas for the soleus thick filament xiii activation has a more graded response. Lastly, x-ray data collected at different lengths demonstrated that the effect of length on soleus is more pronounce compared to EDL, particularly noticeable in the thick filament during relaxation phase after stimulation ceased. These observations indicate that soleus is more length-dependent than EDL. These different approaches to thick filament regulation in fast- and slow-twitch muscles may be designed to allow for short duration, strong contractions versus sustained finely controlled contractions, respectively.
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Title: Biophysical and Computational Characterization of CinDel Edits of Dystrophin
Creator: Stojkovic, Vladimir
Date: 2022
Description: Duchenne muscular dystrophy (DMD) is a degenerative genetic disease caused by a genetic defect that results in the absence of dystrophin, a...
Show moreDuchenne muscular dystrophy (DMD) is a degenerative genetic disease caused by a genetic defect that results in the absence of dystrophin, a protein with an important stabilizing role in muscle cells. DMD causes progressive muscle degeneration leading to the loss of ambulation, and typically results in death before the third decade of life. Treatments for DMD aim to restore dystrophin expression and typically do so by producing edited or modified dystrophins. The only FDA approved therapy, exon skipping, produces dystrophin edits at exon boundaries but emerging therapeutic approaches like gene replacement therapy and CRISPR-Cas9-based gene editing techniques like CinDel allow for greater flexibility and are not constrained to exon boundary edits. However, understanding of what makes a “good”, functional edit is limited so it is not clear how to make use of this increased flexibility to produce optimal edits which are believed to be necessary for robust treatment. In an effort to improve understanding of the biophysics of these non-exon edits, we have embarked on a mixed experimental and computational study of a set of CinDel edits in the D19-D21 region of the dystrophin central rod domain. First, we have conducted an Alphafold structure prediction-based screen of a subset of possible edits in this region and selected one edit for follow-up characterization. We then compared this computationally-selected edit to three other heuristically designed edits experimentally and computationally by molecular dynamics simulations. We found that the computationally selected edit is significantly more thermodynamically stable than the other edits in the cohort. This edit also generally exhibited more favorable properties in MD simulations across multiple measures such as helicity, STR-junction unwinding and conformational variability.
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Title: STRUCTURE AND DYNAMICS OF MODIFIED NUCLEOSOMES UNDER EPIGENETIC REGULATION
Creator: Kohestani, Havva
Date: 2022
Description: Epigenetic regulations are critical in inducing heritable phenotype changes in biological systems without alternating their core genetic DNA...
Show moreEpigenetic regulations are critical in inducing heritable phenotype changes in biological systems without alternating their core genetic DNA sequences. In vivo, reversible epigenetic mechanisms engage various molecular structures from RNAs to larger proteins. The present thesis investigates the influence of epigenetic regulatory factors such as histone protein variants and small non-coding RNAs on the dynamics and structure of nucleosome core particles. Our results show that a histone substitution is an efficient tool in increasing or decreasing the exposure of DNA to post-translational modification (PTMs) factors or larger molecular assembly elements. Substitution of canonical H2A with H2A.B alters DNA-dimer interface resulting in increased breathing and accessibility of DNA. Replacement of canonical H3 with CENP-A variant impacts the overall core-DNA dynamics with flexibility of DNA entry/ exit sites and more rigid tetramer structure. Histone substitution also affects the micro to macro level molecular communication in the nucleosome system. The long-range correlated motions are weakened in H2A.B compared to canonical NCP. We observed a reduction in effective long-range DNA-DNA and DNA-core allosteric pathways in CENP-A NCP compared to canonical and Widom NCPs. Non-coding RNAs increase the tendency of the H3 tail histones to interact with DNA and induce the structural changes in the initial ideal B-DNA of NCP. Overall, the interaction of epigenetic regulatory factors in the form of protein or nucleic acids shifts the energetic and structural properties of the original nucleosome system. As a result, the chromatin structure is prepared to generate the proper biological response throughout spermatogenesis, chromosome segregation, or PTMs assembly.
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