MEMBRANE LOCATION OF SPIN-LABELED M13 MAJOR COAT PROTEIN MUTANTS DETERMINED BY PARAMAGNETIC RELAXATION AGENTS

Mutants of the M13 bacteriophage major coat protein containing single cysteine replacements (A25C, V31C, T36C, G38C, T46C, and A49C) in the hydrophobic and C-terminal domains were purified from viable phage. Th ese were used for site-directed spin-labeling to determine the locatio n and assembly of the major coat protein incorporated in bilayer membr anes of dioleoylphosphatidylcholine. The membrane location of the spin -labeled cysteine residues was studied with molecular oxygen and Ni2ions as paramagnetic relaxation agents preferentially confined to the hydrophobic and aqueous regions, respectively, by using progressive-sa turation electron spin resonance (ESR) spectroscopy. The section of th e protein around Thr36 is situated at the center of the membrane, Resi due Thr46 is placed at the membrane surface in the phospholipid head g roup region with a short C-terminal section, including Ala49, extendin g into the aqueous phase. Residue Ala25 is then positioned consistentl y in the head group region of the apposing lipid monolayer leaflet, Th ese positional assignments are consistent with the observed mobilities of the spin-labeled groups. The outer hyperfine splittings in the ESR spectra decrease from the N-terminal to the C-terminal of the hydroph obic section (residues 25-46), and then drop abruptly in the aqueous p hase (residue 49). Additionally, the strong immobilization and low oxy gen accessibility of residue 25 are attributed to steric restriction a t the hinge region between the transmembrane and N-terminal amphipathi c helices, Sequence-specific modulations of the ESR parameters are als o observed, Relatively low oxygen accessibilities in the hydrophobic r egion suggest intermolecular associations of the transmembrane helices , in agreement with saturation transfer ESR studies of the overall pro tein mobility. Relaxation enhancements additionally reveal a Ni2+ bind ing site in the N-terminal domain that is consistent with a surface or ientation of the amphipathic helix.