University of Illinois Urbana-Champaign

Application of NMR spectroscopy to interrogate the structures of bacterial protein toxin membrane localization domains and amphotericin B

Hisao, Grant Shigeto

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HISAO-DISSERTATION-2017.pdf

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https://hdl.handle.net/2142/97566

Description

Title
Application of NMR spectroscopy to interrogate the structures of bacterial protein toxin membrane localization domains and amphotericin B
Author(s)
Hisao, Grant Shigeto
Issue Date
2017-04-14
Director of Research (if dissertation) or Advisor (if thesis)
Rienstra, Chad M.
Doctoral Committee Chair(s)
Rienstra, Chad M.
Committee Member(s)
  • Burke, Martin D.
  • Sweedler, Jonathan V.
  • Wilson, Brenda A.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Date of Ingest
2017-08-10T19:51:54Z
Keyword(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).
Graduation Semester
2017-05
Type of Resource
text
Permalink
http://hdl.handle.net/2142/97566
Copyright and License Information
Copyright 2017 Grant Shigeto Hisao

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