Exploring structural dynamics of a membrane protein by combining bioorthogonal chemistry and cysteine mutagenesis.

ABSTRACT: The functional mechanisms of membrane proteins are extensively investigated with cysteine mutagenesis. To complement cysteine-based approaches, we engineered a membrane protein with thiol-independent crosslinkable groups using azidohomoalanine (AHA), a non-canonical methionine analogue containing an azide group that can selectively react with cycloalkynes through a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. We demonstrate that AHA can be readily incorporated into the Shaker Kv channel in place of methionine residues and modified with azide-reactive alkyne probes in Xenopus oocytes. Using voltage-clamp fluorometry, we show that AHA incorporation permits site-specific fluorescent labeling to track voltage-dependent conformational changes similar to cysteine-based met

SUBMITTER: Gupta K 

PROVIDER: S-EPMC6850778 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature