Structural and functional studies of bacterial outer membrane proteins
Abstract
This thesis studies two particular bacterial outer membrane proteins called OmpC and
Wzi, focusing on their expression, purification, crystallization and X-ray structure
determination.
A series of four naturally occurring OmpC mutants were isolated from a single patient
with an E. coli infection of liver cysts. The isolated E. coli strains progressively exhibited
increasing breadth of antibiotic resistance in which OmpC was predicted to take a partial
role. We carried out an assay in which a strain of E. coli lacking OmpC was used to
express the first (antibiotic sensitive) and the last (antibiotic resistant) of the clinical
OmpC mutants and drug permeation assessed. Single channel conductance measurements
were carried out and the X-ray structures for all the isolates were determined. Protein
stability was assessed. With these data we propose that changes in the transverse electric
field, not the pore size, underlie the clinically observed resistance to the antibiotics. This
is the first demonstration of this strategy for antibiotic resistance.
Wzi is a novel outer membrane protein involved in the biosynthesis and translocation
mechanism of the K30 antigen from E. coli. The mechanism is a complicated process that
requires several proteins including outer and inner membrane proteins. The protein Wzi
was expressed, purified and crystallized. Initial crystals were tested and diffracted to 15Å. After optimization, a crystal diffracting to 2.4Å has been obtained.
Type
Thesis, PhD Doctor of Philosophy
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