Pinning down the mechanism of transport: probing the structure and function of transporters using cysteine crosslinking and site-specific labelling.

Mulligan, Christopher and Mindell, Joseph A (2017) Pinning down the mechanism of transport: probing the structure and function of transporters using cysteine crosslinking and site-specific labelling. In: Ziegler, Christine, ed. Methods in Enzymology: A Structure-Function Toolbox for Membrane Transporter and Channels. Methods in Enzymology, 594 . Elsevier, pp. 165-202. ISBN 978-0-12-812353-9. (doi:10.1016/bs.mie.2017.05.012) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided)

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https://doi.org/10.1016/bs.mie.2017.05.012

Abstract

Transporters are crucial in a number of cellular functions, including nutrient uptake, cell signaling, and toxin removal. As such, transporters are important drug targets and their malfunction is related to several disease states. Treating transporter-related diseases and developing pharmaceuticals targeting transporters require an understanding of their mechanism. Achieving a detailed understanding of transporter mechanism depends on an integrative approach involving structural and computational approaches as well as biochemical and biophysical methodologies. Many of the elements of this toolkit exploit the unique and useful chemistry of the amino acid cysteine. Cysteine offers researchers a specific molecular handle with which to precisely modify the protein, which enables the introduction of biophysical probes to assess ligand binding and the conformational ensemble of the transporter, to topologically map transporters and validate structural models, and to assess essential conformational changes. Here, we summarize several uses for cysteine-based labeling and cross-linking in the pursuit of understanding transporter mechanism, the common cysteine-reactive reagents used to probe transporter mechanism, and strategies that can be used to confirm cysteine cross-link formation. In addition, we provide methodological considerations for each approach and a detailed procedure for the cross-linking of introduced cysteines, and a simple screening method to assess cross-link formation.

Item Type: Book section
DOI/Identification number: 10.1016/bs.mie.2017.05.012
Uncontrolled keywords: Conformation; Cross-linking; Cysteine; Liposome; Membrane topology; Proteoliposome; Transporter.
Subjects: Q Science > Q Science (General)
Q Science > QP Physiology (Living systems) > QP517 Biochemistry
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Christopher Mulligan
Date Deposited: 08 Aug 2017 08:55 UTC
Last Modified: 29 May 2019 19:17 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/62578 (The current URI for this page, for reference purposes)
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