Probing Structure and Dynamics of Transmembrane Alpha Helices of the S21 Pinholin Protein using Electron Paramagnetic Resonance Spectroscopy

Abstract

The final step of the bacteriophage infection cycle is cell death through membrane lysis. The mechanism of lysis uses a small membrane protein called holin and a muralytic enzyme called endolysin. The holin protein of this study is the S21 pinholin, a small hole forming membrane protein comprised of two transmembrane α-helical domains, TMD1 and TMD2. Only TMD2 is required for membrane hole formation, whereas TMD1 acts as the active inhibitory domain of TMD2. Therefore, in the mechanism of hole formation TMD1 must externalize from the cell membrane. Although the function of TMD2 is well characterized, a lack of information exists regarding TMD1. It is currently believed that TMD1 serves no functionality once externalized from the membrane. However, there is literary evidence suggesting otherwise. It is hypothesized that externalized TMD1s interact with each other using the glycine zipper packing motif to inhibit any TMD1s from looping over to close off the holin hole once formed. These stabilizing interactions could aid in hole formation by stabilizing conformational changes of internal TMD2.This study will be the first time solid phase peptide synthesis (SPPS) is used to create the S21 pinholin protein. The pinholin will be site-specifically spin labeled with MTSL and purified using RP-HPLC. Next will be to determine the degree to which TMD1 is externalized through lipid system incorporation and CW-EPR techniques, followed by confirmation of TMD1 α-helical structure using Circular Dichroism. The TMD1 interactions will be probed using Pulsed EPR DEER technique.Additionally, the local secondary structure of the pinholin's α-helices will be probed using a newly developed deuterated amino acid substituted Pulsed EPR - ESEEM technique. This will be the first application of the technique to a real protein system.