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  • Probing Conformational States of a Target Protein in Escherichia coli Cells by in vivo Cysteine Cross-linking Coupled with Proteolytic Gel Analysis.

Probing Conformational States of a Target Protein in Escherichia coli Cells by in vivo Cysteine Cross-linking Coupled with Proteolytic Gel Analysis.

Bio-protocol (2019-06-20)
Sujeet Kumar, Natividad Ruiz
ABSTRACT

Transporters are dynamic membrane proteins that are essential to the physiology of cells. To function, transporters must cycle between various conformational states, so to understand their mechanistic details, it is critical to characterize how their structure changes during the transport cycle. One approach to studying the dynamics of transporters takes advantage of the chemistry of cysteine by using sulfhydryl-reactive, bi-functional cross-linkers to probe changes in the distance between two specific residues that have been substituted to cysteine. This approach is mostly used to study transporters in vitro, not in their natural cellular environment. Here we describe a protocol based on structure-guided cysteine cross-linking and proteolysis-coupled gel analysis to probe conformational changes of a target transporter in live Escherichia coli cells. Although cross-linking approaches have been used to probe the proximity between transmembrane segments in membrane proteins in vivo, to our knowledge this protocol is the first to be used to interrogate transporter dynamics in cells. The use of this protocol is optimal for proteins with known or modeled structures to guide the replacement of specific residues with cysteines and the selection of cross-linking agents with various spacer arm lengths. This protocol allows for discriminating easily cross-linked and uncross-linked species and does not require the often difficult or unavailable reconstitution of transport activity in an in vitro system. In addition, this protocol could be used to probe the conformation of transporters in cells treated with transport inhibitors in order to better understand their mechanism of action, and potentially dynamic interactions between domains in proteins that are not transporters.

MATERIALS
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Product Description

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