Abstract
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(21) protein of phage ϕ21 is the prototype. Here we report the first in vitro synthesis of the wildtype form of the S(21) pinholin, S(21)68, and negative-dominant mutant form, S(21)IRS, 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](222)/[θ](208) 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. (31)P solid-state NMR spectroscopic data on pinholin indicate a strong interaction of both forms of the pinholin with the membrane headgroups. The (31)P NMR data has an axially symmetric line shape which is consistent with lamellar phase proteoliposomes lipid mimetics.