Expression and structural characterization of the p8 major coat protein from B5 filamentous bacteriophage

• Main Author: Bo, Htet Ei
• Language: English
• Published: University of British Columbia 2011
• Online Access: http://hdl.handle.net/2429/36378

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.