Membrane proteins are essential in biological process for the survival of living organisms. The bacteriophage infection cycle ends cell death through membrane lysis. The precise timing of infected host cell lysis is crucial for the phage life cycle. A small membrane protein called holin plays is crucial to define the time length of phage infection cycle. A decent understanding of the holin protein will help us fight antibiotic resistance. The holin protein of this study is the S21 pinholin, and it is encoded by the S21 gene of phage φ21. It is a small (nano-scale) hole forming membrane protein comprised of two transmembrane alpha-helical domains, TMD1 and TMD2. It forms heptameric holes by oligomerization and reorientation of TMD2 within seconds of its triggering. 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. The unique tool we have been using to study the protein is electron paramagnetic resonance spectroscopy (EPR), that detects species with unpaired electrons. We applied EPR to spin-labeled pinholin mutants associated with mimetic membrane environment to obtain information about the proteins’ structural and dynamic information. Furthermore, the circular dichroism, an absorption spectroscopy method based on the differential absorption of circularly polarized light, is adopted to help us confirm the alpha-helical secondary structure the protein maintains. Combined the experimental data, we can propose the tentative models for both the active and inactive form of the protein. Our research of studying the structural properties via EPR will provide a helpful guide for future studies of pinholin as well as the use of EPR to study other proteins.
Author: Tianyan Li
Faculty Advisor: Gary Lorigan, Department of Chemistry and Biochemistry
Graduate Student Advisor: Tanbir Ahammad, Department of Chemistry and Biochemistry
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