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Biophysical Characterization of an ABC L-methionine Transporter

Citation

Li, Qi Wen (2017) Biophysical Characterization of an ABC L-methionine Transporter. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9125QN9. http://resolver.caltech.edu/CaltechTHESIS:04022017-212739773

Abstract

The ATP-binding cassette (ABC) superfamily is pivotal to a number of important biochemical processes and ubiquitous in all kingdoms of life. Previous studies of ABC transporters have been heavily focused on the structural determination of the different intermediates of the transport cycle. In order to characterize the mechanism of an E. coli L-methionine transporter, which is an ABC importer, we first collated previously reported structural information on the conformational states of several well characterized ABC importers and associated binding proteins, and identified four major conformations (i.e., pre-T, outward, post-T, and inward state). We stabilized these intermediates using appropriate mutations, substrates, and nucleotides. We then studied the kinetics and thermodynamics of the formation of these states using surface plasmon resonance (BiaCore, GE Healthcare) and MicroScale Thermophoresis (NanoTemper). We developed a quantitative model that details the kinetic and molecular mechanism of E. coli MetNI. Towards this goal, we extended the Two-State, alternating access model to include other intermediates that are crucial to transport and are using this to provide a temporal understanding of transport. While this model is developed to describe the behavior of the Lmethionine MetNI importer, it may also have predictive power for other ABC Type I importers, since the NBD’s response for coupling transport to ATP-binding and hydrolysis are highly conserved in this family.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:ATP-binding cassette (ABC), L-methionine Transporter, Biophysical Characterization
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Rees, Douglas C.
Thesis Committee:
  • Gray, Harry B. (chair)
  • Goentoro, Lea A.
  • Chan, David C.
  • Rees, Douglas C.
Defense Date:12 January 2017
Non-Caltech Author Email:qiwenlisyracuse (AT) gmail.com
Funders:
Funding AgencyGrant Number
NSF Graduate Research FellowshipUNSPECIFIED
Record Number:CaltechTHESIS:04022017-212739773
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:04022017-212739773
DOI:10.7907/Z9125QN9
Related URLs:
URLURL TypeDescription
https://doi.org/10.1515/hsz-2015-0131DOIArticle adapted for part of Ch. 3.
ORCID:
AuthorORCID
Li, Qi Wen0000-0001-9493-2316
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10125
Collection:CaltechTHESIS
Deposited By: Qi Li
Deposited On:09 May 2017 22:46
Last Modified:22 Feb 2018 22:24

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