Solid phase synthesis and spectroscopic characterization of the active and inactive forms of bacteriophage S pinholin protein.

The mechanism for the lysis pathway of double-stranded DNA bacteriophages involves a small hole-forming class of membrane proteins, the holins. This study focuses on a poorly characterized class of holins, the pinholin, of which the S protein of phage ϕ21 is the prototype. Here we report the first in vitro synthesis of the wildtype form of the S pinholin, S68, and negative-dominant mutant form, SIRS, both prepared using solid phase peptide synthesis and studied using biophysical techniques. Both forms of the pinholin were labeled with a nitroxide spin label and successfully incorporated into both bicelles and multilamellar vesicles which are membrane mimetic systems. Circular dichroism revealed the two forms were both >80% alpha helical, in agreement with the predictions based on the literature. The molar ellipticity ratio [θ]/[θ] for both forms of the pinholin was 1.4, suggesting a coiled-coil tertiary structure in the bilayer consistent with the proposed oligomerization step in models for the mechanism of hole formation. P solid-state NMR spectroscopic data on pinholin indicate a strong interaction of both forms of the pinholin with the membrane headgroups. The P NMR data has an axially symmetric line shape which is consistent with lamellar phase proteoliposomes lipid mimetics.