Probing The Active And Inactive Forms Of The Bacteriophage S21 Pinholin Protein System Using Magnetic Resonance Spectroscopy

The mechanism for the lysis pathway of double-stranded DNA bacteriophages involves a small hole-forming class of membrane proteins known as the holins. This study focuses on a poorly characterized class of holins, the pinholins, of which the S21 protein of phage ϕ21 is the prototype. This study will show the first in vitro synthesis of the active wildtype form of the S21 pinholin, S2168, and the nonfunctional form, S21IRS, using solid phase peptide synthesis. Site directed spin labeling of the pinholin with the nitroxide spin label MTSL allows for the use of both continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopy to study the pinholin structure and dynamics. CW-EPR results confirm successful incorporation of the pinholin into proteoliposomes, while 3-pulsed ESEEM EPR spectroscopy in tandem with circular dichroism (CD) both confirm proper helical secondary structure. Calculated CD molar ellipticity ratios of both active and inactive forms of the pinholin suggest helical interactions occurring within the bilayer which is consistent with the proposed oligomerization step in models for the mechanism of hole formation in the inner bacterial membrane. Finally, 2H and 31P solid-state nuclear magnetic resonance (NMR) spectroscopy were utilized to probe the interactions of wildtype active S2168 and nonfunctional S21IRS pinholin with the lipid bilayer.