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Title:Application of NMR spectroscopy to interrogate the structures of bacterial protein toxin membrane localization domains and amphotericin B
Author(s):Hisao, Grant Shigeto
Director of Research:Rienstra, Chad M
Doctoral Committee Chair(s):Rienstra, Chad M
Doctoral Committee Member(s):Burke, Martin D; Sweedler, Jonathan V; Wilson, Brenda A
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Nuclear magnetic resonance (NMR) spectroscopy
Bacterial toxins
Membrane localization domains
Amphotericin B
Abstract:Infectious disease is a significant health concern. The transmissible nature of these diseases as well as developing issues such as antimicrobial resistances provide a unique challenge to public health and can have significant and potentially unpredictable impacts to the world economy. Combatting and preventing potential pandemics require a multifaceted approach including the development of new antimicrobials and vaccines as well as in further understanding the interactions between pathogens and hosts. Reported here are studies that investigated infectious diseases from two separate aspects using nuclear magnetic resonance (NMR) spectroscopy. The first aspect investigated the structures of homologous membrane localization domains (MLD) of two different bacterial toxins. The MLDs studied were from the C-terminal domain of the AB toxin Pasteurella multocida toxin (PMT), which is expressed by Pasteurella multocida, and the Ras/Rap1-specific endopeptidase domain of the multifunctional autoprocessing repeats-in-toxins (MARTX) toxin expressed by Vibrio vulnificus. The function of these MLDs is to localize the toxin to membranes, which aid in the targeting of the catalytic domains. The study reveals that these two toxins form 4-helix-bundle motifs in solution, consistent with X-ray crystal structures of homologous bacterial toxin constructs. The second set of studies addresses the antifungal drug amphotericin B (AmB). AmB is a natural product expressed by Streptomyces nodosus and has been used for over fifty years as an antifungal drug. Through its long use, it has evaded antimicrobial resistance. However, because of its high toxicity to humans, it has only been used in life threatening infections. We first developed a protocol to express and purify large quantities of isotopically 13C-labeled AmB through biosynthesis for solid-state NMR (SSNMR) experiments. Purified materials were prepared for SSNMR experiments, where the secondary and tertiary structure of the aggregated for of AmB was studied. These studies will contribute to efforts to develop a structure-based understanding of AmB function, and ultimately to new analogs of AmB that have an improved therapeutic index (ratio of potency to toxicity).
Issue Date:2017-04-14
Type:Thesis
URI:http://hdl.handle.net/2142/97566
Rights Information:Copyright 2017 Grant Shigeto Hisao
Date Available in IDEALS:2017-08-10
Date Deposited:2017-05


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