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Method Development for the Assignment of NMR Peaks and the Determination of Quaternary Protein Structure using Reductive Methylation.

作者:Pamlea Nicole Brady,
畢業學校:LSU
出版單位:LSU
核准日期:2015-12-17
類型:Electronic Thesis or Dissertation
權限:restricted.I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thes....

英文摘要

The work described herein is centered on reductive methylation, a chemical modification reaction, which allows for the introduction of methyl groups at the primary amines of the lysine side chain and at the N-terminus. The resulting methyl groups are effective probes for the study of protein structure and dynamics using nuclear magnetic resonance spectroscopy (NMR). Described herein is work conducted on the development of tools for the study of protein structure using reductive methylation, highlighting options for addressing limitations of the labeling method encountered by its practical use.
Chapter 1 serves as a brief introduction to protein structure and the modification of protein structure, both biological and chemical. This chapter sets the stage for the remaining chapters by establishing the importance of structural studies and how the reductive methylation reaction has been useful to this end. Chapter 2 details work done towards the development of a method to make assignments of NMR peaks of a reductively methylated protein. The method described focuses on addressing the issue of degenerate labeling associated with the reaction, which results in similar degrees of methylation at the reaction sites. The method presented involves the introduction of solid chromatographic media into the reaction and using substoichiometric concentrations of the modifying reagents in order to introduce more variation in the labeling of the reaction sites. The overall objective of this project is to investigate if the introduction of the media and the change in reagent concentration would help to overcome the degenerate labeling and enable more definitive peak assignments. In chapter 3, methods were developed by modifying the sample environments to achieve more resolved and intense NMR peaks of reductively methylated protein. The improvement of resolution and intensity aids peak fitting for accurate peak area calculations, which are used in the determination of percent labeling at each dimethylamine peak in the NMR spectra. Chapter 4 details a study where the overall objective is to investigate the effects of reductive methylation on the determination of protein concentration by the Bradford and Bicinchoninic acid (BCA) assays. The development of a novel use of reductive methylation coupled with paramagnetic relaxation enhancement (PRE) NMR spectroscopy is detailed in Chapter 5 for the determination of the quaternary structural complex of a cysteine protease, papain, and a cysteine protease inhibitor. Chapter 6 will discuss the overall conclusions of this dissertation research and future directions of the work presented.


committee_member - Spivak, David

committee_member - Murray, Kermit

committee_member - Green, Christopher

committee_member - Gilman, Samuel D.

committee_member - Garno, Jayne C.

chair - Macnaughtan, Megan A.


 

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