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 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.

Using unnatural amino acids to selectively label proteins for cellular imaging: a cell biologist viewpoint

“…One approach to accomplish this task is to use a modified AA that resembles a natural one, thus enabling its recognition by the endogenous tRNA and tRNA synthetase. This approach has been demonstrated using methionine analogs [4][5][6][7][8] and was successfully used to visualize newly synthesized proteins in mammalian cells [5][6][7]. However, this approach is not protein-specific, as all endogenous methionine residues can potentially be substituted by the analogous unAA and, therefore, its applicability for the fluorescence imaging of specific proteins is limited.…”

“…Genetic code expansion-based labeling has proven successful for the imaging of several receptors and channels in live cells [4,20,24,32,34] (Fig. 2).…”

“…Genetic code expansion-based intra-and extracellular labeling was also used in photomanipulation techniques, such as FRET and FRAP [4,30,33,34,36,38] ( Fig. 2A).…”

“…2A). Single-molecule FRET approaches were recently developed and applied for resolving the conformational changes of hemagglutinins on virus surfaces and of shaker Kv channels upon activation in Xenopus oocytes [4,39]. Such high-resolution experiments have only been made possible due to the site-selective GCE-based labeling.…”

The current chemical biology tool box for studying ion channels

“…A recent study elegantly demonstrated how ncAAs can also be incorporated by endogenous tRNA/aaRS pairs, i.e. azidohomoalanine via the endogenous Met tRNA/aaRS pair (Gupta et al 2019). Alternatively, it is possible to simultaneously follow conformational changes from Cys-reactive labels and fluorescent ncAAs, enabling to map both extra-and intra-cellular conformational landscapes (Kalstrup & Blunck, 2013.…”