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Structure, Dynamics, and Distance Measurements in Membrane Proteins and Peptides using EPR Spectroscopic Techniques

Ghimire, Harishchandra

Abstract Details

2010, Doctor of Philosophy, Miami University, Chemistry and Biochemistry.
EPR spectroscopic techniques provide powerful methods to study the structural dynamics, topology, and distance measurements of peptides/proteins in membranes and solutions. Cholesterol containing bicelles were studied using X- and Q-band EPR spectroscopy. Cholestane-bicelle EPR spectra were better aligned at Q-band than X-band yielding hyperfine splittings closer to the rigid limit values and resulting in higher order parameters. Increasing cholesterol content in bicelles resulted in lower membrane fluidity and a higher phase transition temperature. Three site-specifically labeled PLB samples were studied using aligned EPR spectroscopy. The transmembrane helical tilt of WT-PLB was determined to be (13 ± 4)°. EPR line-shape analysis revealed highly restricted motion in transmembrane domain of PLB. Both the loop and the cytoplasmic domain were found to have two distinct conformational states, which agrees with previous studies in the literature. The major T-conformer interacts with membrane with slower dynamics, and the minor R-conformer has faster dynamics with minimal membrane interaction. The distance and orientation between two TOAC nitroxides on AChR M2δ were determined using aligned EPR spectroscopy coupled with rigorous spectral simulations. An internitroxide distance of 14.6 A was obtained from a dual-labeled peptide, matching closely with modeling results (14.5 A). The orientational angles of the two nitroxides were also determined from simulations, which compared favorably to the molecular studies. DEER spectroscopy was used to measure the distance between two monomers in a GCN4-LZ dimer using TOAC spin labels. We observed a 13-fold increase in sensitivity by collecting DEER spectra at Q-band when compared to X-band. This increased sensitivity represented a 169-fold decrease in data collection time and revealed a greatly improved frequency spectrum with higher quality distance data. Three GCN4-LZ peptides and one Aβ-40 peptide were synthesized, purified, and characterized. One of the GCN4-LZ peptides was used to measure the inter-monomer distance and the binding force in a dimer. The Aβ-40 peptide was used to study the differential association of apolipoprotein ε-3 and ε-4 isoforms with its oligomer in solution. A protocol was developed for the optimal peptide synthesis with TOAC spin labeling.
Gary A. Lorigan, PhD (Advisor)
Michael W. Crowder, PhD (Committee Chair)
Ann E. Hagerman, PhD (Committee Member)
John W. Hawes, PhD (Committee Member)
Luis A. Actis, PhD (Committee Member)
120 p.

Recommended Citations

Citations

  • Ghimire, H. (2010). Structure, Dynamics, and Distance Measurements in Membrane Proteins and Peptides using EPR Spectroscopic Techniques [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1291739688

    APA Style (7th edition)

  • Ghimire, Harishchandra. Structure, Dynamics, and Distance Measurements in Membrane Proteins and Peptides using EPR Spectroscopic Techniques. 2010. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1291739688.

    MLA Style (8th edition)

  • Ghimire, Harishchandra. "Structure, Dynamics, and Distance Measurements in Membrane Proteins and Peptides using EPR Spectroscopic Techniques." Doctoral dissertation, Miami University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=miami1291739688

    Chicago Manual of Style (17th edition)