Structure-function relationship of viral coat proteins : a site-directed spectroscopic study of M13 coat protein

This thesis describes the results of a spectroscopic study of the major coat protein of bacteriophage M13. During the infection process this protein is incorporated into the cytoplasmic membrane of Escherichia coli host cells. To specifically monitor the local structural changes and changes in the environment of the protein upon membrane insertion, a set of cysteine site-specific mutants of protein was produced for the purpose of ESR spin labeling and fluorescence spectroscopy. These spectroscopic techniques, in combination with CD spectroscopy, are particularly suitable for comparison of protein structural changes in different membrane model systems. The spectroscopic experiments indicate that the very tight structure of the phage particle was disrupted only with strong ionic detergents, such as SDS and CTAB. However the phage structure was not affected by either lipids or nonionic detergents. On the other hand, after a chloroform-induced transformation of the filamentous phage particle into the S- form, the major coat protein was completely solubilized under these conditions. Upon solubilization of the phage particle in sodium cholate at low pH, a protein "structural dimer" appeared to be the most stable aggregate. This structural dimer, in which the protein subunits that are slightly shifted with respect to each other, is proposed to play a key role in the assembly and disassembly of the phage particle in vivo. However, when completely solubilized in the membrane, the major coat protein is stable in a monomer state, and does not have a tendency to aggregate. Site-directed ESR spin labeling was found to be a useful technique to compare the protein structure and topology in micellar and lipid bilayer model systems. To allow a stable association with different membrane model systems, the local structure of the major coat protein changed significantly, but surprisingly the major structural elements, such as the α-helix content, are largely retained. The detailed topology of the major coat protein in lipid bilayers was determined by using Ni 2+quenchers with the spin-labeled mutants. The results show that the part of the major coat protein around amino acid residue Thr36 is situated in the centre of the membrane. Amino acid residues 25 and 46 are located in the lipid head group region at the two water-membrane interfaces, with only a short part of the C-terminus (three to four amino acid residues) extending into the aqueous phase. This transmembrane topology leaves the α-carbons of Lys40, Lys43, and Lys44 in the membrane interior, while the ε-amino groups of the lysine side chains probably interact with the large excess of phosphates in the lipid head groups. Since the major coat protein has to aggregate to form a protective coat around viral DNA, these results indicate that lipids should be removed first at the assembly site, before the major coat protein can interact with DNA to form a new virus coat.