Chapter 9. Methanethiosulfonate Reagent Accessibility Studies, Cysteine-Scanning Mutagenesis, Protein Overexpression, and Functional Reconstitution: A Strategy for Studying the Structure/Function Relationships within the Mitochondrial Citrate Transport Protein

  1. Michael W. Quick Ph.D.
  1. Ronald S. Kaplan

Published Online: 19 MAR 2003

DOI: 10.1002/0471434043.ch9

Transmembrane Transporters

Transmembrane Transporters

How to Cite

Kaplan, R. S. (2002) Methanethiosulfonate Reagent Accessibility Studies, Cysteine-Scanning Mutagenesis, Protein Overexpression, and Functional Reconstitution: A Strategy for Studying the Structure/Function Relationships within the Mitochondrial Citrate Transport Protein, in Transmembrane Transporters (ed M. W. Quick), John Wiley & Sons, Inc., Hoboken, NJ, USA. doi: 10.1002/0471434043.ch9

Editor Information

  1. Department of Neurobiology, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA

Author Information

  1. Department of Biochemistry and Molecular Biology, Finch University of Health Sciences, The Chicago Medical School, North Chicago, IL, USA

Publication History

  1. Published Online: 19 MAR 2003
  2. Published Print: 23 AUG 2002

Book Series:

  1. Receptor Biochemistry and Methodology

Book Series Editors:

  1. David R. Sibley

Series Editor Information

  1. Molecular Neuropharmacology Section, Experimental Therapeutics Branch, NINDS, National Institutes of Health, Bethesda, Maryland, USA

ISBN Information

Print ISBN: 9780471065135

Online ISBN: 9780471434047

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Keywords:

  • mitochondria;
  • citrate;
  • transporter;
  • methanethiosulfonate;
  • cysteine-scanning;
  • overexpression;
  • liposomes

Summary

An approach is described in which cysteine scanning mutagenesis is combined with overexpression and functional reconstitution of transporter mutants, followed by a determination of the accessibility of engineered transmembrane cysteine residues to methanethiosulfonate reagents. This strategy enables an identification of water-accessible and -inaccessible residue locations within a transmembrane domain, and provides inferential information regarding the secondary structure of the domain and the residues that likely comprise a portion of the aqueous translocation pathway through a given transporter. We have used this approach to elucidate the structure/function relationships within the mitochondrial citrate transport protein. Experimental protocols and related issues are discussed in detail.