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

Abstract

During infection the major coat protein of the filamentous bacteriophage M13 is in the cytoplasmic membrane of the host Escherichia coli. This study focuses 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 protein mutants used are successfully produced in mg quantities by overexpression in E. coli. Mutant coat proteins are labeled and reconstituted into mixed bilayers of phospholipids. Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. Additional information is obtained by determining the accessibility of the fluorescence quenchers acrylamide and 5-doxyl stearic acid. By employing uniform coat protein surroundings provided by TFE and SDS, local effects of the backbone of the coat proteins or polarity of the residues could be excluded. Our data suggest that at a lipid to protein ratio around 100, the N-terminal arm of the protein gradually enters the membrane from residue 3 towards residue 19. The hinge region (residues 17-24), connecting 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 acid residues lysine 8, phenylalanine 11 and leucine 14, results in a rearrangement of the N-terminal protein part into a more extended conformation. The 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 protein ratio. The influence of the phospholipid headgroup composition on the rearrangement of the N-terminal part of the protein is found to be negligible within the range thought to be relevant in vivo. From our experiments we conclude that membrane-anchoring and space-limiting effects are key factors for the structural rearrangement of the N-terminal protein part of the coat protein in the membrane.

Ultrafast resonance energy transfer from a site-specifically attached fluorescent chromophore reveals the folding of the N-terminal domain of CP29

“…Concentrations of pigments were determined spectroscopically; Chls as described by Porra et al [29] and Cars as described by Davies [30]. CP29 apoprotein mutants and wild-type were labeled with MTS-TAMRA, (2-((5(6)-tetramethylrhodamine)carboxylamino)ethyl methanethiosulfonate from TRC, Toronto, Canada), and purified from the excess of TAMRA using HPSEC and affinity chromatography on a His-Trap column [31]. Reconstitution and purification of protein-pigment complexes were performed as reported in [15].…”

Anchoring mechanisms of membrane-associated M13 major coat protein

“…1) (Koehorst et al, 2004;Meijer et al, 2001a;Spruijt et al, 2000;Spruijt et al, 2004;Vos et al, 2005). There seems to be a weak point in the helix structure between the N-terminal and the transmembrane helix, the so-called hinge region, which allows the amphipathic part of the protein to be optimally reconstituted in the lipid bilayer.…”

“…The amphipathic and the hydrophobic transmembrane part contain the most important hydrophobic anchoring elements. In addition there are specific aromatic and charged amino acid residues in these domains of the coat protein that fine-tune the association of the protein to the membrane (Koehorst et al, 2004;Meijer et al, 2001a,b;Spruijt et al, 2000Spruijt et al, , 2004Stopar et al, 2003). A remarkable feature of the major coat protein in vivo is its ability to enter and leave the membrane (Brissette and Russel, 1990;Dunker et al, 1991;Griffith et al, 1981;Guy-Caffey et al, 1992;Webster, 1983, 1985;Makowski, 1992;Manning et al, 1981Manning et al, , 1982.…”

Membrane-bound Conformation of M13 Major Coat Protein

“…The mutants were purified and labeled with 1,5-I-AEDANS (purchased from Molecular Probes) as described previously (15). Labeled coat protein was reconstituted in DOPC/DOPG (80/20 molar ratio, purchased from Avanti Polar Lipids) using the cholate dialysis method (16).…”