| Summary: | This thesis is based on work done on the expression, purification and structural
characterization of the major coat protein of B5 bacteriophage. The major coat proteins of
bacteriophages have long been utilized to understand membrane proteins and membrane-associated assembly. It is the special feature of the major coat protein to exist in different
environments that holds the key to its involvement in phage assembly. The structure of p8 in
the different environments, especially in the host membrane, has to be fully understood
before the mystery of phage assembly can be solved. The major coat protein of B5, p8, has
been chosen in this study because B5 infects Gram positive bacteria and the structure of p8 in
an appropriate model membrane can better represent its native structure in the host
membrane.
In Chapter 1, I introduce background information on filamentous phage, and the
debate of major coat protein structure. The different structures that already exist for the major
coat protein in virion, in host membranes, and during phage assembly are discussed.
In the next chapter, I present the steps required to obtain pure p8 using a heterologous
bacterial expression system. The optimizations and considerations needed to express and
purify p8 are discussed thoroughly. The considerations taken for p8 expression can
essentially be applied to other membrane protein expression. In the same chapter, an I32C
mutant of p8 is also designed, expressed and successfully purified. The technique used to
introduce the single substitution mutation to p8 can be applied to other protein mutation
experiments.
In the subsequent chapter, p8 structure is studied using circular dichroism (CD),
nuclear magnetic resonance (NMR) and site directed labeling with a 6-bromoacetyl-2-
dimethylaminonaphthalene (BADAN) fluorescence probe. The results from CD show that p8
has high alpha helicity when reconstituted into lipid compositions that represent the Gram
positive membrane. Preliminary NMR experiments have been performed and conditions to
obtain optimal NMR spectra have been explored. BADAN fluorescence labeling experiments
have been trialed and have been shown to successfully indicate the local environment of
residue 32 to which BADAN is attached. Finally, possible future work is discussed.
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