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
- Michael W. Quick Ph.D.
Published Online: 19 MAR 2003
Copyright © 2002 by Wiley-Liss, Inc.
How to Cite
Kaplan, R. S. (2003) 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
Department of Neurobiology, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA
- Published Online: 19 MAR 2003
- Published Print: 23 AUG 2002
Book Series Editors:
- David R. Sibley
Series Editor Information
Molecular Neuropharmacology Section, Experimental Therapeutics Branch, NINDS, National Institutes of Health, Bethesda, Maryland, USA
Print ISBN: 9780471065135
Online ISBN: 9780471434047
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.