Localization and rearrangement modulation of the N-terminal arm of the membrane-bound major coat protein of bacteriophage M13

During infection the major coat protein of the filamentous bacteriophage M1 3 is in the cytoplasmic membrane of the host Escherichia coli. This study f ocuses on the configurational properties of the N-terminal part of the coat protein in the membrane-bound state. For this purpose X-Cys substitutions are generated at coat protein positions 3, 7, 9, 10, 11, 12, 13, 14, 15, 17 , 19, 21, 22, 23 and 24, covering the N-terminal protein part. All coat pro tein mutants used are successfully produced in mg quantities by overexpress ion in E. coli. Mutant coat proteins are labeled and reconstituted into mix ed bilayers of phospholipids. Information about the polarity of the local e nvironment around the labeled sites is deduced from the wavelength of maxim um emission using AEDANS attached to the SH groups of the cysteines as a fl uorescent probe. Additional information is obtained by determining the acce ssibility of the fluorescence quenchers acrylamide and 5-doxyl stearic acid . By employing uniform coat protein surroundings provided by TFE and SDS, l ocal effects of the backbone of the coat proteins or polarity of the residu es could be excluded. Our data suggest that at a lipid to protein ratio aro und 100, the N-terminal arm of the protein gradually enters the membrane fr om residue 3 towards residue 19. The hinge region (residues 17-24), connect ing the helical parts of the coat protein, is found to be more embedded in the membrane. Substitution of one or more of the membrane-anchoring amino a cid residues lysine 8, phenylalanine 11 and leucine 14, results in a rearra ngement of the N-terminal protein part into a more extended conformation. T he N-terminal arm can also be forced in this conformation by allowing less space per coat protein at the membrane surface by decreasing the lipid to p rotein ratio. The influence of the phospholipid headgroup composition on th e rearrangement of the N-terminal part of the protein is found to be neglig ible within the range thought to be relevant in vivo. From our experiments we conclude that membrane-anchoring and space-limiting effects are key fact ors for the structural rearrangement of the N-terminal protein part of the coat protein in the membrane. (C) 2000 Elsevier Science B.V. All rights res erved.