Experimental, Computational and, Theoretical Analysis of the GroEL/GroES Catalytic Cycle

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dc.contributor.advisorLorimer, George Hen_US
dc.contributor.authorCorsepius, Nicholas Craneen_US
dc.contributor.departmentBiochemistryen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2015-02-06T06:35:40Z
dc.date.available2015-02-06T06:35:40Z
dc.date.issued2014en_US
dc.description.abstractThe GroEL/GroES system is among the best characterized molecular chaperonins, having been the focus of numerous studies since its discovery just over 40 years ago. There are several aspects of the chaperonin's catalytic cycle that have been thoroughly established, such as the positive intra-ring and negative inter-ring cooperativity that characterizes the binding of ATP, or the concerted domain movements within a ring that constitute the T → R allosteric transition. However, there are still several aspects of the GroEL/GroES catalytic cycle that are still not well understood, and the focus of this work is to address several of them. The results presented throughout this dissertation differ, sometimes drastically, from conventional wisdom, providing a new framework for the investigation of GroEL/GroES catalysis. The work presented here contains several new insights into the GroEL/GroES catalytic cycle. The complicated nature of the interaction between GroEL and its substrate proteins (SPs) has previously prevented quantitative analysis of the role that SP binding plays in the cycle. The problem is circumvented in this work by application of an SP surrogate that simultaneously mimics the effects of bound SP and allows for the quantification of allosteric states. Development of the system leads to the first ever quantification of the role of SP in the catalytic cycling of GroEL. In addition to cycle features that had not been previously addressed, there are several parts of the catalytic cycle that have simply been overlooked throughout the years or unduly simplified in previous analysis. The hydrolysis of ATP within a ring and the release of ADP from a ring are often characterized as concerted events. Both steps are crucial to the cycling of the GroEL/GroES system, and are thoroughly investigated in this work. Thorough examination reveals that neither step is well-characterized as a concerted process. Pre-steady state kinetic analysis demonstrates that hydrolysis of ATP occurs stochastically, each subunit hydrolyzing ATP independently and randomly. Pre-steady state kinetic analysis also reveals that ADP release is a multi-step process. Insight into the steps preceding/ accompanying ADP release are found in molecular dynamics (MD) simulations of the R → T allosteric relaxation. These simulations demonstrate the allosteric relaxation proceeds through a highly asymmetric series of states. Furthermore, the relaxation path taken by subunits can be described as exhibiting dynamic negative cooperativity. The analysis implicates the allosteric relaxation as the trigger that allows the GroEL/GroES system to effectively respond to environmental stimuli. The last topic addressed concerns the role of the GroEL/GroES2 symmetric complex and the mechanism governing its cycling. It has long been assumed that a ring must hydrolyze all seven ATP before it can discharge GroES. This assumption is challenged by new insight into the stochastic mechanism of ATP hydrolysis. The analysis presented in this paper undermines the idea of all-or-none cycling and builds a quantitative relationship between the nucleotide distribution in a complex and its propensity to dissociate.en_US
dc.identifierhttps://doi.org/10.13016/M2G895
dc.identifier.urihttp://hdl.handle.net/1903/16157
dc.language.isoenen_US
dc.subject.pqcontrolledBiochemistryen_US
dc.subject.pqcontrolledPhysical chemistryen_US
dc.subject.pqcontrolledBiophysicsen_US
dc.subject.pquncontrolledallosteric relaxationen_US
dc.subject.pquncontrolledasymmetryen_US
dc.subject.pquncontrolledbreakage of symmetryen_US
dc.subject.pquncontrolledfootballen_US
dc.subject.pquncontrolledstochastic hydrolysisen_US
dc.subject.pquncontrolledTMR dimeren_US
dc.titleExperimental, Computational and, Theoretical Analysis of the GroEL/GroES Catalytic Cycleen_US
dc.typeDissertationen_US

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