Spin-label ESR of bacteriophage M13 coat protein in mixed lipid bilayers. Characterization of molecular selectivity of charged phospholipids for the bacteriophage M13 coat protein in lipid bilayers

Abstract

Bacteriophage M 13 major coat protein has been incorporated at different lipid/protein ratios in lipid bilayers consisting of various ratios of dimyristoylphosphatidylcholine (DMPC) to dimyristoylphosphatidylglycerol (DMPG). Spin-label ESR experiments were performed with phospholipids labeled at the C-14 position of the sn-2 chain. For M 13 coat protein recombinants with DMPC alone, the relative association constants were determined for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels and found to be 1 .O, 1 .O, and 2.1 relative to the background DMPC, respectively. The number of association sites for each phospholipid on the protein was found to be 4 per protein monomer. The intrinsic off-rates for lipid exchange at the intramembranous surface of the protein in DMPC alone a t 30 OC were found to be 5 X lo6, 6 X lo6, and 2 X lo6 s-' for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels, respectively. Adding DMPG to the DMPC lipid system increased the exchange rates of the lipids on and off the protein. By gel filtration chromatography, it is found that protein aggregation is reduced after addition of DMPG to the lipid system. This is in agreement with measurements of tryptophan fluorescence, which show a decrease in quenching efficiency after introduction of DMPG in the lipid system. The results are interpreted in terms of a model relating the ESR data to the size of the protein-lipid aggregates.%e nonenveloped filamentous bacteriophage M 13, which is closely related to bacteriophages fd and fl (Ray, 1977), enters the Escherichia coli cell, leaving its major coat protein (a product of gene 8) in the cytoplasmic membrane (Marvin & Wachtel, 1975). During DNA replication and synthesis of new phage proteins, the coat protein is stored in the membrane as an integral membrane protein. Newly synthesized coat protein, which arrives at the membrane as a preprotein and is converted to the mature coat protein by the action of a leader peptidase (Kiihn et al., 1986), is stored in the membrane and encapsulates the viral DNA molecule during the membrane-associated assembly of new virus particles. The parental coat protein, after it is incorporated in the membrane, is also used for the formation of new infectious M13 bacteriophages (Armstrong et al., 1983). The coat protein of M13, whose amino acid sequence is known (Van Wezenbeek et al., 1980;Beck et al., 1978), consists of three different regions: a basic carboxyl terminus, a central hydrophobic core of 19 amino acids, and an acidic amino terminus (Chamberlain et al., 1978; Van Wezenbeek et al., 1980). Spin-label electron spin resonance (ESR)' has been shown to be a particularly useful technique for studying the interactions of M13 coat protein in reconstituted lipid systems (Datema et al., 1987a). Three groups of phospholipids could be distinguished showing different specificities for the M 13 coat protein (group I, CL and PA; group 11, PG, PS, and SA; and group 111, PC and PE). These spin-label expe...

Integration of a K+ Channel-Associated Peptide in a Lipid Bilayer: Conformation, Lipid-Protein Interactions, and Rotational Diffusion

“…Judging from the large values of the effective rotational times obtained from the STESR spectra of the peptide labeled with 5-InVSL, it seems likely, whatever the exact form of the enclosed j3-sheet structures of the K26(L18C) peptide is, that these are also assembled in oligomeric aggregates of what are possibly pore structures. Consistency with the models proposed above for the stoichiometry of the motionally restricted lipid associated with the K26 peptide would require that some of the motionally restricted lipids be trapped within the oligomeric structures, a feature that is exhibited by the lipid-bound M13 phage coat protein in its @-sheet form (Wolfs et al, 1989).…”

“…The ESR spectra of the spinlabeled lipids indicate that the K26 peptide assemblies present a well-defined hydrophobic surface in the bilayer at which the lipid chains are restricted in their rotational mobility. It has been observed previously that the M13 phage coat protein incorporated in lipid bilayers in a polymeric @-sheet conformation gives rise to lipid spin label ESR spectra in which the motionally restricted component is much better resolved than in those arising from the protein incorporated in a mainly a-helical conformation (Peelen et al, 1992;Wolfs et al, 1989). The reason for this spectral difference lies partly in the exchange rate of the lipids at the protein interface, which is rather slow in the case of the polymeric P-sheet structure.…”

Quantification of protein–lipid selectivity using FRET

“…The problem of lipid selectivity by the M13 MCP has been extensively addressed through electron spin resonance (ESR) techniques (Wolfs et al. 1989; Peelen et al. 1992), and selectivity constants were recovered for the interaction of the protein with different phospholipids.…”

“…However, due to MCP aggregation, only five annular binding sites were identified by protein molecule when using ESR (Wolfs et al. 1989). Optimization of the protein purification procedure allowed monomeric MCP to be obtained after membrane incorporation (Spruijt et al.…”

A small protein in model membranes: a time-resolved fluorescence and ESR study on the interaction of M13 coat protein with lipid bilayers

“…The/~-polymeric form is an aggregated/%sheet protein complex (Datema et al 1987;Wolfs et al 1989;Spruijt and Hemminga 1991;Van Gorkom et al 1990), whereas the protein in the e-oligomeric form has a high amount of e-helix structure and forms only small aggregates, in dynamic equilibrium with protein monomers (Spruijt et al 1989;Spruijt and Hemminga 1991). It has been argued by Spruijt and Hemminga (1991) that this form is the biologically active state of the protein.…”