Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Pharmacology

First Advisor

William F. DeGrado

Second Advisor

Mark D. Goulian

Abstract

Two-component systems represent the dominant mechanism for cellular signal transduction in prokaryotes. One particular system, PhoQ, has been the focus of our interest because it is the switch that controls virulence in Salmonella and other pathogenic gram negative bacteria. Certain domains of PhoQ and other two-component systems have been studied extensively and this research has yielded several structures with atomic level resolution. However, no complete structure or experimentally-based model has been forthcoming and the precise mechanism by which these diverse systems transmit signals from the exterior of the cell to the interior has remained elusive. We have undertaken a study to examine the E coli PhoQ from a structural and mechanistic viewpoint. We discovered a transmembrane polar residue that is conserved among many two-component systems that is critical for signaling in PhoQ. Critically, this feature is shared between PhoQ and the only two-component protein for which a transmembrane domain structure has been solved at the atomic level, HtrII. We undertook a disulfide scanning mutagenesis experiment to probe the structure of the transmembrane domain of PhoQ. We found evidence that PhoQ shares a similar topology with HtrII, especially the presence of a hemi-channel--a striking feature characterized by a group of tightly packed helices that sharply diverge at one end of the bundle to form a pocket that allows water molecules to penetrate into the core of the protein. We used the inferred homology between HtrII and PhoQ to create a model of the TM domain of PhoQ. Through statistical analysis of our Cys-crosslinking data, we determined that our data represented the average of different conformations of PhoQ brought about by changes in signaling state. We determined that the data was best explained by two models of PhoQ, which represent independent signaling states. This discovery represents the first evidence of a two-state model of activation of a Two-component protein, which we present alongside the first full-length model of a sensor kinase.

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