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Probing the structural dynamics, conformational change, and topology of pinholin S21, a bacteriophage lytic protein, using electron paramagnetic resonance spectroscopy

Ahammad, Tanbir
http://orcid.org/0000-0003-0000-9702
http://rave.ohiolink.edu/etdc/view?acc_num=miami1595598100557068

Abstract Details

2020, Doctor of Philosophy, Miami University, Chemistry and Biochemistry.
Bacteriophages have evolved an efficient, protein-mediated host cell lysis mechanism that terminates the infection cycle and facilitates the release of progeny virions at an optimal time. Among the lytic proteins, holin controls the first and rate-limiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time and concentration. Recently, a prototype holin called pinholin has been reported which makes nanoscale holes that are too small for the passage of endolysin. However, the holes formed by pinholin dissipate the cytoplasmic membrane potential leading to the release and activation of membrane-tethered signal anchor release endolysins which are already exported to the periplasm. Pinholin is the evolutionary ancestor of holin, but was discovered more recently and, as such, has not been fully studied in the literature. Of all the pinholin systems, S21 from lambdoid phage 21 is one of the most well-known. Pinholin S21 consists of two holin proteins: the active pinholin S2168 and the inactive antipinholin S2168IRS. Each of these proteins have a short N-terminal domain followed by two transmembrane domains connected by a short loop and terminate in a long, positively charged C-terminal region. However, the precise structural details of the proteins in this system is not well understood. The works presented in this dissertation were carried out to structurally characterize the proteins of pinholin S21 using biophysical techniques including state-of-the-art electron paramagnetic resonance (EPR) spectroscopic methods. Continuous Wave (CW) EPR line shape analysis and power saturation (PS) experiments showed that both transmembrane domains (TMDs) of S2168IRS had restricted mobility meaning they were incorporated in the lipid bilayer while the termini regions had higher mobility as they were solvent exposed. However, TMD1 of S2168 was found to be partially externalized from and interacting with surface of the lipid bilayer while TMD2 remained inside of the lipid bilayer. DEER spectroscopy was used for detailed structural studies and direct comparison between the two forms of pinholin S21. These experiments validated and refined the topology and structural models of both pinholin proteins. The effects of residue mutations on the structural topology and conformational changes of pinholin S21 were also probed using CW-EPR PS and DEER spectroscopic techniques. These studies expanded the application of EPR spectroscopic techniques for the study of membrane proteins in general while also providing a deeper understanding of the structural dynamics, conformational changes, and topology of the complex pinholin S21 protein system.
Gary Lorigan, Ph.D. (Advisor)
Carole Dabney-Smith, Ph.D. (Committee Chair)
Rick Page, Ph.D. (Committee Member)
David Tierney, Ph.D. (Committee Member)
Paul Urayama, Ph.D. (Committee Member)
181 p.
Biochemistry; Biophysics; Chemistry
Holin; Pinholin; EPR; DEER; Bacteriophage;

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Citations

  • Ahammad, T. (2020). Probing the structural dynamics, conformational change, and topology of pinholin S21, a bacteriophage lytic protein, using electron paramagnetic resonance spectroscopy [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1595598100557068

    APA Style (7th edition)

  • Ahammad, Tanbir. Probing the structural dynamics, conformational change, and topology of pinholin S21, a bacteriophage lytic protein, using electron paramagnetic resonance spectroscopy. 2020. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1595598100557068.

    MLA Style (8th edition)

  • Ahammad, Tanbir. "Probing the structural dynamics, conformational change, and topology of pinholin S21, a bacteriophage lytic protein, using electron paramagnetic resonance spectroscopy." Doctoral dissertation, Miami University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=miami1595598100557068

    Chicago Manual of Style (17th edition)

miami1595598100557068
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This open access ETD is published by Miami University and OhioLINK.