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

1247-Pos Board B198 Probing Structure and Dynamics of Externalized Transmembrane Alpha Helices of S21 Pinholin Protein using Electron Paramagnetic Resonance Spectroscopy Daniel Drew. Chemistry and Biochemistry, Miami University, Oxford, OH, USA. The last step of the bacteriophage infection cycle is cell death through lysis, the breakdown of the cell membrane. The mechanism of lysis uses two proteins, a small membrane protein called the 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 a-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 be externalized from the cell membrane. Although the function of TMD2 is well characterized there is a lack of information regarding TMD1. It is currently believed that TMD1 serves no functionality once externalized from the membrane. However, there is substantial literary evidence suggesting otherwise. It is hypothesized that the externalized TMD1s interact with each other using the glycine zipper a-helical packing motif to inhibit any TMD1s from looping over to close off or block the holin hole once formed. These interactions between TMD1s could also stabilize the pinhole formation and conformational changes of TMD2 inside the membrane. This study will be the first time solid phase peptide synthesis (SPPS) will be used to make the S21 pinholin protein. The holin proteins will be sitespecifically spin labeled with MTSL and the purity will be confirmed using MALDI-TOFmass spectroscopy. Next will be to determine the degree to which TMD1 is externalized through lipid system incorporation and EPR power saturation techniques. This is followed by confirmation of TMD1 a-helical structure retention post externalization. Finally, externalized TMD1 interactions will be probed using DEER and ESEEM EPR techniques to measure distances between neighboring TMD1s.