Theses and Dissertations from UMD
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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 Experimental, Computational and, Theoretical Analysis of the GroEL/GroES Catalytic Cycle(2014) Corsepius, Nicholas Crane; Lorimer, George H; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The 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.