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- W2985714717 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 methods. By combining AHA incorporation and cysteine mutagenesis in an orthogonal manner, we were able to site-specifically label the Shaker Kv channel with two different fluorophores simultaneously. Our results identify a facile and straightforward approach for chemical modification of membrane proteins with bioorthogonal chemistry to explore their structure-function relationships in live cells." @default.
- W2985714717 created "2019-11-22" @default.
- W2985714717 creator A5001712498 @default.
- W2985714717 creator A5020708402 @default.
- W2985714717 creator A5081586330 @default.
- W2985714717 date "2019-11-12" @default.
- W2985714717 modified "2023-09-23" @default.
- W2985714717 title "Exploring structural dynamics of a membrane protein by combining bioorthogonal chemistry and cysteine mutagenesis" @default.
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