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.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Mulligan C,Mindell JAdoi
10.1016/bs.mie.2017.05.012subject
Has Abstractpub_date
2017-01-01 00:00:00pages
165-202eissn
0076-6879issn
1557-7988pii
S0076-6879(17)30155-6journal_volume
594pub_type
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