BIOPHYSICAL STUDIES OF THE PF1 COAT PROTEIN IN THE FILAMENTOUS PHAGE, IN DETERGENT MICELLES, AND IN A MEMBRANE ENVIRONMENT

During the assembly of the Pf1 phage, the membrane-bound coat proteins convert into subunits of the filamentous phage. Fourier-transform infrared (FT-IR) transmission spectroscopy has been applied to a study of the secondary structure of these coat proteins when present (a) in the phage, (b) in detergent micelles, and (c) in a phospholipid membrane aqueous system. Suspensions of the Pf1 phage in H2O and (H2O)-H-2 show an amide I band at 1652 cm-1, indicative of a high content of the alpha-helical structure present. Oriented films of the Pf1 phage studied by polarized FT-IR transmission spectroscopy indicate that the alpha-helical structures as well as the tyrosine residues (band at 1515 cm-1) are both aligned along the axis of the phage. When the Pf1 coat protein is present in sodium dodecyl sulfate detergent micelles and in lipid membrane systems, the FT-IR spectra show an amide I band at 1657-1658 cm-1, also indicative of a predominantly alpha-helical secondary structure. H/H-2 amide proton exchange studies show that when present in a phospholipid membrane system some 50-60% of the Pf1 protein exchanges rapidly, while the rest undergoes slow exchange. This is consistent with a proportion of the protein being exposed to the solvent and the other being embedded in the lipid bilayer. The presence of a band at 1630-1640 cm-1 is indicative of the presence of random structures. Oriented lipid films containing the Pf1 coat protein studied by internal reflectance polarized FT-IR spectroscopy indicate that the average alpha-helical structures of the Pf1 coat protein are oriented perpendicular to the plane of the lipid membrane. To establish whether the amino-terminal region penetrates the lipid bilayer and perturbs the lipid chains, a fluorescence polarization study was carried out using the probe diphenylhexatriene. These studies and additional calorimetric studies of the gel-liquid-phase transition of the lipid in protein/lipid systems are consistent with a perturbation expected only from a single helix spanning the lipid bilayer.