Date of Award

Spring 1-1-2015

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry & Biochemistry

First Advisor

Joseph J. Falke

Second Advisor

Robert T. Batey

Third Advisor

Johannes Rudolph

Abstract

CheA is a multi-domain histidine kinase believed to be a central component of all bacterial and archaeal chemosensory arrays. The best studied CheA proteins are those of Escherichia coli, Salmonella typhimurium, and Thermotoga maritima. The P4 catalytic domain of CheA is of particular interest due to its role in ATP binding and auto-phosphorylation of the substrate His residue on the P1 substrate domain, a process which involves Mg2+ in the P4 domain ATP-binding pocket. CheA auto-phosphorylation reaction has also long been known to be sensitive to the monovalent cation composition of the reaction buffer. Recent studies in GHKL superfamily members MutL (from E coli) and BCK (from Rattus norvegicus) demonstrated improved activity in the presence of monovalent K+. Here experimental and computational analyses assess the effect of monovalent cations on the activity of Salmonella typhimurium CheA, and generate a model for a putative monovalent cation binding pocket. The findings show that CheA prefers the physiological K+ ion over the other monovalent cations tested, indicating the site is specific and tuned to possess the appropriate K+ affinity for the cytoplasmic environment.

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