|Title||Membrane-anchoring interactions of bacteriophage major coat proteins|
|Source||Wageningen University. Promotor(en): T.J. Schaafsma; M.A. Hemminga. - S.l. : S.n. - ISBN 9789058084071 - 102|
|Publication type||Dissertation, internally prepared|
|Keyword(s)||manteleiwitten - laserfluorescentiespectroscopie - paramagnetische elektronenresonantiespectroscopie - bacteriofagen - coat proteins - laser fluorescence spectroscopy - electron paramagnetic resonance spectroscopy - bacteriophages|
The major coat proteins of the filamentous bacteriophages Pf3 and M13 are stored in the inner membrane of the cell during the reproductive cycle. In this process, protein-lipid anchoring interactions are important for the formation of the correct structure of these proteins in the membrane, enabling fast and efficient phage assembly. The focus of this thesis is on the role of domains and specific amino acids on the position of these proteins in the membrane. Both proteins are studied using site-specific probing using fluorescence and ESR spectroscopy. This biophysical approach provides information about the relative depth and dynamics of specific sites of the protein in the membrane. In previous structural views of the membrane-bound state of the M13 coat protein, the protein is thought to be in an L-shape, in which the N-terminal arm of the protein is positioned along the membrane surface. The spectroscopic studies described in this thesis show that the amphipathic N-terminus of the protein is not firmly associated with the membrane surface, but is also present in a more extended configuration. It is demonstrated that the hydrophobic amino acids in the N-terminal arm play an important role in the topology of the helix at the membrane surface. Furthermore, it is found that at the C-terminal side of the helical transmembrane domain, the charged lysines and hydrophobic phenylalanines are involved in strong anchoring interactions with the membrane interface region. These amino acids affect the location of the entire helical transmembrane domain. The results of a site-specific probing study on the Pf3 major coat protein reveals striking similarities with those obtained from the M13 major coat protein. Both proteins exhibit a strong structural coherence, in spite of the low primary sequence homology. Therefore, the tertiary structure of the Pf3 major coat protein should closely resemble the membrane-bound structure of the M13 coat protein. Apparently, domains of amino acids, which are comparable in physico-chemical characteristics, but lack sequence homology, are able to assemble in a structural coherent manner. In conclusion, the relatively simple coat proteins are positioned in the membrane via a complicated set of protein-lipid interactions provided by individual amino acids as well as domains of amino acids.