Browsing by Subject "Biophysics"

Now showing 1 - 4 of 4
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    Biophysical Methods to Study Bromodomain Interactions and 19F Nmr Analysis of Cbp/P300 Kix Domain Complexes
    (2020-01) Ycas, Peter
    Epigenetics is the study of heritable changes in genome expression which alter organism phenotype. Genome expression is controlled by access to specific DNA, which in turn is controlled by how DNA associates with the histone proteins it is wrapped around. The condensed complex of DNA and histones is known as a chromosome. A hallmark of epigenetics is post-translational modification of chromosomes, both of DNA and histones. One such post-translational modification is acetylation of lysines on histone tails, a modification associated with gene transcription. The first part of this thesis focuses on bromodomain and plant homeodomain (PHD) finger transcription factor containing protein (BPTF), a member of the bromodomain class of proteins. Bromodomains recognize histone lysine acetylation and recruit transcription factors and nucleosome remodelers to chromatin. BPTF is a multi-domain protein which is the largest part of the nucleosome remodeling factor. The second half of this thesis examines another transcriptional regulator, the KIX domain of CBP/p300, a protein domain which co-localizes transcription factors. In Chapter one, the role of BPTF in gene expression and disease progression is discussed. The success of using small molecule probes to study other bromodomain containing proteins is described, highlighting the need to develop probes for BPTF in order to further discern the role of the protein in healthy and disease states. The current advances in small molecule inhibitor and biophysical tool development are described. The second half of Chapter one introduces the role of the KIX domain of CBP/p300 and its’ interaction partners and focuses on the past methods of studying the domain with a variety of NMR techniques. Chapter two describes the development and ligand deconstruction analysis of the first BPTF bromodomain inhibitor, AU1. The structure activity relationship of the molecule is developed using protein-observed fluorine NMR (PrOF NMR) and the (S) enantiomer of the compound is identified as the active component. Treatment of a panel of cancer cell lines which were found to be BPTF dependent through gene knockdowns showed decreased cell viability when treated with AU1. Development of biophysical tools to rigorously characterize high affinity BPTF inhibitors is described in Chapter three. The progress of ligand development in Chapter two was hampered by the lack of tools to accurately determine tight binding affinities. To address these issues, SPR and AlphaScreen assays were developed which are capable of rank ordering compounds which previously could not be done with PrOF NMR. These biophysical assays were validated against a small panel of previously characterized small molecule inhibitors of BPTF. Following validation, these assays were used to discover two new BPTF scaffolds which were explored as possible starting points for BPTF ligand discovery. Finally, the development of conditions to co-crystallize ligands with BPTF and the solution to their X-ray structure is described providing BPTF-small molecule ligand structural information for the first time. Chapter four compares fluorine labeling strategies of the KIX domain of CBP/p300 with 2-, and 3-fluorotyrosine (2FY and 3FY). The response of small molecule mimics of five fluorinated aromatic amino acids to changes in solvent polarity is used as a barometer for their utility in PrOF NMR. The chemical shift anisotropy of polycrystalline samples of 2FY and 3FY are determined by magic angle spinning. Through incorporation of both fluorinated tyrosine derivatives into the KIX domain of CBP/p300, their influence on stability and pKa perturbation in a model protein is investigated. The response of both 19F labeling strategies to ligand binding is discussed using the protein-protein interaction partner, MLL, which shows an allosteric response with 2FY KIX which is not observed with 3FY KIX. Chapter five delves into the differences observed with 2FY and 3FY KIX. The 19F NMR response of both proteins to a positive allosteric regulator (MLL), a negative allosteric regulator (naphthol AS-E phosphate), and an as yet unknown small molecule (2) are measured with changes in D2O solvent composition to measure the solvent accessibility of 19F nuclei upon ligand binding. The role of aromatic amino acids in the allosteric communication network is interrogated through ternary complex formation with MLL and c-Myb, which bind concurrently at separate sites on KIX. Finally, efforts towards crystallizing a stabilized complex of 2FY and 3FY KIX to discern their differences in allosteric communication are described.
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    Bridging the gap between theory, experiments and simulations of nanochannel confined DNA
    (2020-08) Bhandari, Aditya Bikram
    The study of nanochannel confined DNA has garnered substantial attention since the early 2000's owing to its application in genome mapping, the coarse-grained counterpart to DNA sequencing, which is an indispensable tool in biological research. However, our understanding of the physics behind confined DNA is rather simplified and incomplete. Thus, theory, simulation and experiment have by and large been at odds with one another. The results of this dissertation are aimed at understanding and attempting to resolve the source of these discrepancies. Our strategy for this dissertation is three-pronged. First, we revisit a historically cited explanation for the discrepancies - the lack of understanding behind the wall depletion length denoting the wall-DNA electrostatic interactions. Second, we considered the intersection of theory and simulation, which recent developments have managed to bring sufficiently into accord. We found that the deviations between the fractional extension distributions predicted by an asymptotic theory and those observed experimentally, are not due to a breakdown of the theory, even for experimental conditions which typically do not strictly satisfy the asymptotic limits of the theory. This motivated a closer inspection of the theories to determine a missing link between theory and experiment. Finally, by studying a recently generated dataset of fractional extensions spanning a wide range of the experimental parameter space, we were able to isolate this missing link as the effect of long-range electrostatics in the system which are typically ignored in the simplified theories, wherein the DNA is assumed as a neutral polymer confined in a channel of a reduced effective channel size. We believe that our findings within this dissertation will provide a better understanding of confined polymers and, in particular, the nanochannel confined DNA system used in genome mapping, as well as provide new directions of study in the future.
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    The explicit polarization theory as a quantum mechanical force field and the development of coarse-grained models for simulating crowded systems of many proteins
    (2014-01) Mazack, Michael John Morgan
    This dissertation consists of two parts. The first part concerns the use of explicit polarization theory (X-Pol), the semiempirical polarized molecular orbital (PMO) method, and the dipole preserving, polarization consistent (DPPC) charge model as a quantum mechanical force field (QMFF). A detailed discussion of Hartree-Fock theory and X-Pol is provided, along with expressions for the energy and the analytical first derivative of this QMFF. Test cases for this QMFF with extensive comparisons to experimental data and other models are provided for water (XP3P) and hydrogen fluoride (XPHF), showing that the PMO/X-Pol/DPPC approach discussed in this dissertation is competitive with the most accurate models for those two chemical species over a wide range of chemical and physical properties.The second part of this dissertation concerns the development and application of coarse-grained models for protein dynamics. First, a coarse-grained force field (CGFF) for macromolecules in crowded environments is introduced and described along with a visualization environment for the cartoon-like rendering of biomolecules in vivo. This CGFF is tested against experimental diffusion coefficients for myoglobin (Mb) at a wide range of concentrations, including volume fractions as high as 40%, finding it to be surprisingly accurate for its simplicity and level of coarseness. Second, an analytical coarse-grained (ACG) model for mapping the internal dynamics of proteins into a spherical harmonic expansion is described.
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    Integrated Fluorescence Spectroscopy for FRET Analysis of Novel Ionic Strength Sensors in the Presence of a Hofmeister Series of Salts
    (2019-07) Miller, Robert
    Living eukaryotic cells are complex, crowded, and dynamic organisms that continually respond to environmental and intracellular stimuli. In addition, these cells have heterogeneous ionic strength with compartmentalized variation of both intracellular concentrations and types of ions. The underlying mechanisms associated with ionic strength variations that trigger different biological functions and response to environmental cues remain largely unknown. Therefore, there is a need to develop a quantitative method for mapping the compartmentalized ionic strength and their temporal fluctuations within living cells. In this work, we investigate a class of novel ionic- strength sensors that consists of tethered mCerulean3 (a cyan fluorescent protein) and mCitrine (a yellow fluorescent protein) via a linker of varied amino acids. In these protein constructs, mCerulean3 and mCitrine act as a donor-acceptor pair undergoing fluorescence resonance energy transfer (FRET) based on both the linker amino acids and the environmental ionic strength. The energy transfer efficiency and the donor-acceptor distance of these sensors can be quantified noninvasively using integrated fluorescence methods in response to intracellular ionic strength in living eukaryotic cells. We employed time-resolved fluorescence methods to monitor the excited-state dynamics of the donor in the presence and absence of the acceptor as a function of the environmental ionic strength using potassium chloride (KCl, 0–500 mM). Towards mapping out the response to of these sensors towards biologically relevant salts, we carried out time- resolved fluorescence for FRET analysis of these sensors as a function of the Hofmeister series of salts (KCl, LiCl, NaCl, NaBr, NaI, Na2SO4). We also used these results towards technique development for FRET analysis based on time-resolved fluorescence polarization anisotropy. Our results show that the energy transfer efficiency of these sensors is sensitive to both the linker amino acid sequence and the environmental ionic strength. These studies in a controlled environment complement previous steady-state spectroscopy analysis of these sensors in a cuvette with the advantage of the compatibility of our approach with fluorescence lifetime imaging microscopy on living cells.