LOCAL DYNAMICS OF THE M13 MAJOR COAT PROTEIN IN DIFFERENT MEMBRANE-MIMICKING SYSTEMS

The local environment of the transmembrane and C-terminal domain of M1 3 major coat protein was probed by site-directed ESR spin labeling whe n the protein was introduced into three membrane-mimicking systems, DO PC vesicles, sodium cholate micelles, and SDS micelles. For this purpo se, we have inserted unique cysteine residues at specific positions in the transmembrane and C-terminal region, using site-directed mutagene sis. Seven viable mutants with reasonable yield were harvested: A25C, V31C, T36C, G38C, T46C, A49C, and S50C. The mutant coat proteins were indistinguishable from wild type M13 coat protein with respect to thei r conformational and aggregational properties. The ESR data suggest th at the amino acid positions 25 and 46 of the coat protein in DOPC vesi cles are located close to the membrane-water interface. In this way th e lysines at positions 40, 43, and 44 and the phenylalanines at positi ons 42 and 45 act as hydrophilic and hydrophobic anchors, respectively . The ESR spectra of site specific maleimido spin-labeled mutant coat proteins reconstituted into DOPC vesicles and solubilized in sodium ch olate or SDS indicate that the local dynamics of the major coat protei n is significantly affected by its structural environment (micellar vs bilayer), location (aqueous vs hydrophobic), and lipid/protein ratio. The detergents SDS and sodium cholate sufficiently well solubilize th e major coat protein and largely retain its secondary structure elemen ts. However, the results indicate that they have a poorly defined prot ein-amphiphilic structure and lipid-water interface as compared to bil ayers and thus are not a good substitute for lipid bilayers in biophys ical studies.