Study the Structural Topology, Dynamic Properties and Functional Model of Phage 21 Holin Protein using EPR Spectroscopy

Abstract

Phage mediated bacterial cell lysis is the most frequent cytocidal event in this biosphere and strictly controlled by at least three groups of lytic protein. The holin, a small membrane protein, is the essential part of this lytic system and responsible for the initiation of lytic process by making holes in the inner cytoplasmic membrane at a precise time. The holin of the phage Φ21 makes smaller holes and known as the pinholin S21 (PHS21). Pinholin S21is encoded by the S21 gene. S21 is a dual start motif gene, can be expressed as active pinholin (68 amino acids) or antiholin (71 amino acids). Both pinholin and antiholin have two transmembrane domains (TMD). In the active form, TMD1 can be externalized from the membrane very quickly to oligomerize followed by very small hole formation, but in antiholin this externalization is slow which delay the oligomerization. We are using Electron Paramagnetic resonance (EPR) spectroscopic techniques to probe the structural topology, dynamics properties and functional model of the tactive and inactive forms of PHS21. Fmoc-Solid Phase Peptide Synthesis and site-directed spin labeling (SDSL) was used to make EPR active peptide samples. Samples were incorporated into DMPC proteoliposomes to mimic a membrane environment. EPR line shape analysis, power saturation and Double electron-electron resonance (DEER) spectroscopic data probed the structural topology and dynamic properties of pinholin S21 and demonstrated significant differences between the active and inactive forms of PHS21. EPR spectroscopic data and circular dichroism (CD) spectra indicate the concentration-dependent oligomerization by the active form of PHS21.