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Synthetic methodologies for labeling membrane proteins and studies utilizing electron paramagnetic resonance in biologically relevant lipid architectures

Mayo, Daniel J.
http://rave.ohiolink.edu/etdc/view?acc_num=miami1343434201

Abstract Details

2012, Doctor of Philosophy, Miami University, Chemistry and Biochemistry.
This dissertation illustrates several new methodologies that have been developed to examine and characterize membrane proteins and peptides using spin label EPR spectroscopy. Background on the methodologies used in this dissertation can be found in Chapter 1. The structural topology or orientation of a peptide or protein in a membrane is very important for functional characterization. Two categories of topologies, peripheral and integral, were investigated using two model peptides, magainin 2 and AchR M2δ, using aligned glass plate CW-EPR techniques. Aligning the membrane and the peptide with respect to the magnetic field allows the distinction between these two peptide topologies that cannot easily be determined in an non-oriented spectrum. This mechanical alignment technique allows researchers to quickly determine if a peptide is laying on the surface or buried inside the membrane (Chapter 2). The antimicrobial peptide magainin 2 was further assessed using the same alignment CW-EPR technique. A new methodology using the same techniques was developed to characterize the topology, degree of insertion, secondary structure, and how magainin 2 are oriented on the membrane surface (Chapter 3). Utilizing CW-EPR alignment and a unique bicelle lipid architecture, the topology of the AchR M2δ peptide was further investigated (Chapter 4). The secondary structure of the AchR M2δ peptide was also assessed using EPR spectroscopy. A novel approach was developed to determine the local secondary structure of the transmembrane AchR M2δ peptide using pulsed EPR spectroscopy. Additionally a flexible MTSL spin label was used, which is very amenable to larger membrane and globular proteins (Chapter 5). An effort was thereafter made to address some of the limitations of the approaches described in previous chapters. A major synthetic and biologically challenging project was pursued following work done by the Hubbell and Schultz labs. The rigid unnatural amino acid TOAC was incorporated into two membrane proteins KCNE1 and Tha4 using state-of-the-art molecular biology techniques. For the Tha4 case, E60TOAC was successfully introduced in a cell free wheat germ over-expression system, and a CW-EPR spectrum was obtained (Chapter 6). Concluding remarks and future directions are also provided (Chapter 7).
Gary Lorigan, A. (Advisor)
Michael Crowder, W. (Committee Chair)
Carole Dabney-Smith (Committee Member)
Scott Hartley, C. (Committee Member)
Gary Janssen, R (Committee Member)
109 p.
Biochemistry
EPR; electron paramagnetic resonance; unnatural amino acid incorporation; TOAC; alignment epr; magainin-2; AchR;

Recommended Citations

Citations

  • Mayo, D. J. (2012). Synthetic methodologies for labeling membrane proteins and studies utilizing electron paramagnetic resonance in biologically relevant lipid architectures [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1343434201

    APA Style (7th edition)

  • Mayo, Daniel. Synthetic methodologies for labeling membrane proteins and studies utilizing electron paramagnetic resonance in biologically relevant lipid architectures. 2012. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1343434201.

    MLA Style (8th edition)

  • Mayo, Daniel. "Synthetic methodologies for labeling membrane proteins and studies utilizing electron paramagnetic resonance in biologically relevant lipid architectures." Doctoral dissertation, Miami University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=miami1343434201

    Chicago Manual of Style (17th edition)

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© 2012, all rights reserved.
This open access ETD is published by Miami University and OhioLINK.