Using kinetics to decipher the mechanism of protein transport by the Escherichia coli Sec machinery : a dissertation /
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| Format: | Thesis Book |
| Language: | English |
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[College Station, Tex.] :
[Texas A&M University System Health Science Center],
[2011]
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| Abstract: | ABSTRACT: In Escherichia coli, the majority of newly synthesized transported proteins across or into the cytoplasmic membrane are mediated by the membrane-embedded complex termed "SecYEG translocase". In the post-translational translocation, the molecular chaperone SecB binds the full-length unfolded preprotein, and targets it to the SecYEG-bound SecA. Subsequently, the preprotein is translocated through the SecYEG pore using the energy from ATP hydrolysis by SecA and the proton motive force. The central dogma in the Sec transport field for over 15 years is that SecA is a motor protein, and that ATPase activity results in translocation of a preprotein in discrete steps. The transport kinetics observed with a novel real-time fluorescence-based transport assay with 1 s time resolution allowed us to directly test the predictions of this model. If protein transport through the SecYEG pore is the rate-limiting step of transport, our data suggest that precursor movement through the Sec machinery is not directly driven by a series of SecA conformational cycles. Instead, our current hypothesis is that Brownian motion of the precursor protein is sufficient for it to translocate through the SecYEG pore and SecA ATPase regulates pore accessibility. The influence of the transmembrane electric field gradient on protein export by the Sec machinery had been investigated for over two decades. An obvious implecation of the electric field gradient is that positively charged precursor proteins should transport slower and less efficiently due to electrophoretic effects on charge mobility. We found a strong positional effect of poly-lysine, and that lengthy consecutive lysine segments are more inhibitory than multiple well-distributed, poly-lysine segments. These data support a model in which the local effects of positive charge on the translocation kinetics dominate over total thermodynamic constraints. The translocation times of precursors with multiple positively charged sequences, or "pause sites", were fairly well predicted by a local effect model. However, the kinetic profile predicted by this local effect model was not observed, suggesting that local effects have global consequences, perhaps by influencing abortive transport probability. In total, these data suffest a complex interplay between precursor primary structure, local secondary structure, and the translocation system. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". "Submitted to the Office of Research and Graduate Studies of The Texas A&M University System Health Science Center in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2011" Approved as to style and content by: Siegfried M. Musser, J. Martin Scholtz, Ry Young, Jean-Philippe Pellois, Geoffrey Kapler. |
| Physical Description: | xiv, 188 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 149-153). |