Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item Examining the role of water and hydrophobicity in folding, aggregation, and allostery(2018) Custer, Gregory Scott; Matysiak, Silvina; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Solvation and hydrophobicity drive many critical processes in nature, playing an important role in the folding of proteins, aggregation of surfactants into micelles, and in the disorder to order transitions that occur in some allosteric proteins upon ligand binding. Understanding how solvation and hydrophobicity affect these processes at a molecular level is important to finding new ways to use these processes, but it can be difficult to characterize these molecular details using experimental methods. Molecular dynamics (MD) simulations have proven useful in exploring details and thermodynamic conditions inaccessible in experiment, as MD captures the time evolution of the system at a molecular level. The phenomena which can be studied with an MD simulation depend on the mathematical model employed. Atomistic models provide the most detail for a simulation, but due to the computational costs required are not typically used to study phenomena which require large systems and time scales greater than several μs. Coarse-grained (CG) models reduce the complexity of the system being studied, enabling the exploration of phenomena that occur at longer time scales. We have developed CG models to study protein folding and surfactant aggregation. Our CG surfactant model uses a three-body potential to account for hydrogen bonding without an explicit electrostatic potential, reducing the computational cost of the model. With our surfactant model we studied the stability of non-ionic micelles at extremes of temperature, capturing a window of thermal stability with destabilization of the micelles at both high and low temperatures. We observed changes in structure and solvation of the micelle at low temperatures, with a shift in enthalpy of solvation water providing the driving force for destabilization. Solvation and hydrophobicity are also critical in the folding and stability of proteins. With a modified version of our surfactant model we characterized the folding landscape of a designed sequence which folds to a helical bundle in water. We found two competing folded states which differ by rotation of a helix and trade between hydrophobic packing and solvation of protein's core. Changes in hydrophobic packing can also be involved in the disorder to order transitions that occur upon liganding binding in an allosteric protein, such as the E. Coli biotin ligase/repressor (BirA), in which ligand binding promotes dimerization. We have used atomistic simulations of BirA mutants in collaboration with an experimental group to identify structural changes, accompanied by changes in solvation, at both the dimer interface and ligand binding regions for distal mutations which impact the functionality of BirA.Item Probing Allosteric Communication Between Disordered Surfaces in a Protein(2015) Cressman, William John; Beckett, Dorothy; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Molecular mechanisms of protein allostery are not well understood, particularly in those systems that undergo disorder to order transitions upon activation. BirA, an E.coli metabolic enzyme and transcriptional repressor, is a model allosteric protein in which corepressor, bio-5’-AMP, binding enhances dimerization by -4 kcal/mol and is coupled to disordered loop folding on both functional surfaces. In this work, BirA variants with single alanine substitutions in dimerization surface residues are investigated to further characterize communication between the two sites. Isothermal titration calorimetry (ITC) measurements of corepressor binding of these BirA variants indicate only the G142A substitution perturbs the Gibbs free energy of binding. The G142A crystal structure indicates a mechanism of communication from the corepressor binding to the dimerization surface involving α-helical extension of residues 143-146. Measurements of the heat capacity changes associated with corepressor binding to the BirA variants support a model in which the helical extension enhances dimerization by enabling the formation of a network of intramolecular interactions on the dimerization surface.Item DEVELOPING A NEW MODEL OF THE GROEL FUNCTIONAL CYCLE AND ITS IMPLICATIONS FOR THE GROEL-OPTIMIZED SUBSTRATE PROTEIN REFOLDING(2014) Ye, Xiang; Lorimer, George H; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Despite years of research work, many aspects of the fundamentally important GroEL functional cycle are still in dispute. The work of this dissertation mainly focuses on three major disputes in the field: the identity of the rate determining step (RDS), the physiological order of arrival of ligands (ATP, SP and GroES) to the GroEL trans ring, and the role of the symmetric GroEL-GroES2 "football" complex in the overall chaperonin cycle. With multiple carefully designed spectroscopic probes, a pre-steady state survey has been conducted on the kinetics of the GroEL functional cycle. From the survey, a two cycle model emerges: in the absence of SP, ADP release is the RDS of the asymmetric cycle and consequently, the asymmetric GroEL-GroES1 ,"bullet" which precedes this step, is the pre-dominant species. In this mode, the machine turns over very slowly, minimizing futile ATP consumption. Due to the slow release of ADP, the system turns over in a well defined manner with the two rings operating 180o out of phase of each other, analogous to a two-stroke motor. In the symmetric cycle, which operates in the presence of SP, the release of ADP is greatly accelerated while the intrinsic ATPase activity of GroEL remains unaffected. Consequently ATP hydrolysis becomes the RDS and the symmetric GroEL-GroES2 "football" becomes the predominant species. Contrary to previous chaperonin dogma, the symmetric complex is a highly dynamic species exchanging its two bound ligands, GroES and encapsulated SP from both rings with a half time ~1sec. Switching to a parallel processing machine, the chaperonins turns over rapidly, ultimately driven by stochastic hydrolysis of ATP which causes the symmetric complex to undergo breakage of symmetry (BoS). With such a dynamic system, folding in the `folding cage' seems less important in GroEL-mediated SP refolding as suggested by the passive refolding model. Instead, GroEL may play a more active role in achieving its central biological function as indicated by this two cycle model. This may be the very reason why employing even as low as one GroEL ring per ten SP can achieve SP refolding to a similar extent as using a stoichiometric amount.Item ROLE OF SALT BRIDGES IN GROEL ALLOSTERY(2014) Yang, Dong; Lorimer, George H; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Chaperonin GroEL facilitates protein folding with two stacked back-to-back, identical rings and the "lid", co-chaperonin GroES. The mis-folded/unfolded substrate protein (SP) adjusts the chaperonin cycling from an asymmetric to a symmetric cycle by catalyzing the release of ADP from the trans ring of GroEL, thus promoting the R to T allosteric transition. ATP binding to the SP bound ring promotes the association of a second GroES and subsequently a GroEL-GroES 2 "football" complex is formed as the folding functional form. However, ADP does release spontaneously, albeit at very slow rate, in the absence of SPs. The intrinsic mechanism by which GroEL relaxes to the lower potential energy T state remains poorly understood. A network of salt bridges forms and breaks during the allosteric transitions of GroEL. Residue D83 in the equatorial domain forms an intra-subunit salt bridge with K327 in the apical domain, and R197 in the apical domain forms an inter-subunit salt bridge with E386 in the intermediate domain. These two salt bridges stabilize the T state and break during the T to R state transition. Removal of these salt bridges by mutation destabilizes the T state and favors the R state of GroEL. These mutations do not alter the intrinsic ATPase activity of GroEL. However, the affinity for nucleotides becomes enhanced and ADP release is hindered such that SP cannot displace the equilibrium to the T state, as normally it does in the wild type. The exchange of ADP to ATP and association of a second GroES is compromised with the following GroEL-GroES 2 "football" formation is hindered. These mutations do not completely eliminate the T state, in the absence of nucleotide, as shown biochemically and by crystal structures. The biased allosteric equilibrium hampers the formation of folding active "football" complex as the mutant GroEL's incompetency to revisit T state in the presence of nucleotide, but not due to the elimination of its T state. This study revealed the critical role of salt bridges in regulating the allosteric transitions of GroEL and conjugated formation of the "football" complex.Item Molecular basis of the kinematics of the kinesin step(2012) Zhang, Zhechun; Thirumalai, Devarajan; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Kinesin is an ATP-dependent cellular transporter that ferries cargos towards the plus-end of a microtubule. Despite significant advances in experiments, which have provided deep insights into the motility of kinesin, the molecular events that occur in a single step have not been fully resolved. In order to provide these details, this thesis develops a structure of the complex between kinesin and microtubule, and devises new simulation methods to probe the stepping kinetics over a wide range of conditions. Hundreds of molecular movies of kinesin walking on the microtubule are generated using coarse-grained simulation methods. Analysis of these movies shows that there are three major stages in the stepping kinetics of kinesin. In addition, an allosteric network within kinesin, responsible for controlling nucleotide release, is identified using microsecond all-atom simulations. These simulations are used to answer two important questions. First, does kinesin move by a "power stroke" or by diffusion? During a single step, the trailing head of the kinesin detaches from the microtubule, passes the microtubule-bound leading head, and attaches to the target binding site 16 nm away. The target binding site, however, is one of eight accessible binding sites on the microtubule. Is it possible that the "power stroke" (a large conformational change) in the leading head, pulls the trailing head into the neighborhood of the target binding site? This remained unclear because the fraction of the 16 nm step associated with the power stroke and diffusion had never been quantified. Second, how does the microtubule accelerate ADP release from kinesin, which is a key step in completing a single step? The ADP binding site of kinesin is more than 1.5 nm away from the microtubule binding surface. Therefore, the microtubule must affect the ADP binding site through an allosteric mechanism. However, the structural basis for transmitting signals through the underlying allosteric network was previously unknown. Analysis of hundreds of kinesin steps generated using coarse-grained simulations showed that the power stroke associated with the docking of the neck linker to the leading head, is responsible for only 4 nm of the 16 nm step, and the remaining 12 nm is covered by diffusion. However, the power stroke in the leading head constrains the diffusion of the trailing head, decreases the probability of side steps, and therefore biases the trailing head, to the target binding site. Additional all-atom simulations of the ADP-kinesin-microtubule complex, revealed a surprisingly simple allosteric network within kinesin that explains the acceleration of ADP release upon microtubule binding. The allosteric network also explains two additional experimental observations on ADP release from kinesin.Item Allostery and GroEL: Exploring the Tenets of Nested Cooperativity(2004-06-24) Gresham, Jennifer Suzanne; Lorimer, George H; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Despite a wealth of structural and biochemical studies on the functional cycle of the <i>E. coli</i> chaperonins GroEL and GroES, no model proposed to date accounts for all the effects seen experimentally by the various allosteric ligands: ATP, ADP, SP, GroES, and K+. The work in this dissertation explores the various allosteric transitions in the GroEL reaction cycle and offers a refined model for nested cooperativity that successfully accounts for the effects of these ligands. Initial studies take advantage of a single ring variant, termed SR1, to examine the allosteric properties of GroEL in the absence of complicating interactions arising from negative cooperativity. Initial rates of ATP hydrolysis by GroEL and SR1 as a function of ATP concentration were fit to an equation that makes no arbitrary assumptions. A novel role for K+ and SP is proposed, which suggests they help regulate the negative cooperativity and control the timing of the chaperonin cycle. The kinetics of association of GroES to the trans ring of the asymmetric complex were also studied, using stopped flow fluorescence energy transfer (FRET), revealing that conditions which accelerate dissociation of the cis ligands also accelerate association to the trans ring. This, along with previous work obtained by our lab, suggests that the allosteric signal transmitted between the rings for cis ligand release is the binding of ATP to the T state of the trans ring. A mechanism for the formation of symmetrical particles, termed "footballs," is suggested.