TIP AND TIP CHAPERONE INTERACTION IN YERSINIA AND PSEUDOMONAS

Abstract

Spectroscopy is one of the most wildly used approaches to determine various properties ofproteins. A wide range of spectroscopic methods are available to answer biological questions such as protein structure dynamics and protein-protein interactions at different sample states and timescales. In this dissertation, I have used electron paramagnetic resonance (EPR) spectroscopy to determine the structural dynamics of the tip chaperone proteins of the type III secretion system (T3SS). I have also determined protein-protein and protein-small molecule interactions of T3SS proteins using spectroscopic techniques such as Förster Resonance Energy Transfer (FRET), and nuclear magnetic resonance (NMR). The T3SS macromolecular assemblage found in pathogenic Gram-negative bacteria such as Salmonella typhimurium, Shigella flexneri, Yersinia pestis, and Pseudomonas aeruginosa cause infections by transporting virulence effector proteins that modulate the host cell for the survival and propagation of the pathogens. The structural component of the T3SS is the needle complex which consists of the base, needle, tip complex, and translocon. The tip complex is assembled by multiple copies of the tip protein. In Yersinia and Pseudomonas, prior to the assembly of the needle complex, the tip proteins interact with small cytoplasmic tip protein chaperones LcrG and PcrG to prevent the premature secretion of the tip proteins LcrV and PcrV, respectively. There are no high-resolution structures of tip chaperone proteins. NMR spectroscopy shows LcrG and PcrG as partially folded alpha helices in contrast to the evidence in the literature which predicts the tip protein chaperones to be a coiled-coil. In this dissertation I have reported the different conformations of LcrG and PcrG in the free from and when bound to their cognate tip proteins LcrV and PcrV using EPR spectroscopy.