Using Click Chemistry and Voltage Clamp Fluorimetry to Study Structural Dynamics of Membrane Proteins

Abstract

The conformational dynamics of membrane proteins has been extensively investigated using cysteine mutagenesis to probe accessibility with thiol-reactive compounds and/or introduce fluorophores, spin labels, etc. Here we explored a complementary approach using an unnatural amino acid containing an azide group that can selectively react with alkynes through click chemistry. We found that the azide-containing amino acid can be efficiently introduced into Shaker, a voltage-activated potassium (Kv) channel and allowed us to efficiently tag the protein with alkyne derivatized biotin. The azide containing amino acid could also be labeled with alkyne-conjugated fluorophores to track the conformational transitions of the voltage-sensor activation and deactivation, and the resulting fluorescence voltage (F-V) relationships were very similar to those obtained with fluorophores attached to introduced cysteine residues via thiol-based chemistry. Our results establish the potential of fluorescent labeling through click chemistry to probe the structural rearrangements of membrane proteins, which when combined with cysteine-based labeling should allow two-color labeling of membrane protein assemblies for studying both intramolecular and intermolecular structural dynamics.