Ion channels act as incredible signal integrators throughout the body. They can respond to membrane voltage, extracellular ligands, intracellular ligands, lipids, post-translational modifications, and many protein modulators. All these signals alter the state of the channel, changing the propensity for the channel to open. How this slew of modulators change the conformation of the channel and how those conformational changes are linked to a common gate motivates my work.
We are using multiple techniques to look at conformational dynamics of ion channels. Understanding how a protein works at the molecular level starts with an atomic scale structure. X-ray crystallography and cryoEM provide atomic detail of the overall fold of a protein. From that starting point, we utilize double electron-electron resonance (DEER) spectroscopy and transition metal ion FRET (tmFRET). DEER is an advanced EPR technique that allows us to develop coarse-grain structural models of conformational ensembles in proteins and complexes. DEER can measure distances of up to 8nm between paramagnetic spin labels attached to cysteine residues in a protein of interest. The power of DEER is in its ability to determine conformational equilibria and resolve conformational heterogeneity.
tmFRET is a FRET techniques that uses transition metal ions as FRET acceptors. In doing so, we reduce the effective range over which we can measure FRET to a useful intramolecular range of around 8-25 angstroms.In addition, we decrease the size of the fluorphores from the typical large FPs or dyes to a pair consisting of a small dye and an even smaller metal ion. The combination of these two improvements allows for the accurate measurement of conformational changes within a protein of interest. The strength of this method comes in our ability to measure the FRET associated with a conformational change simultaneously with the readout of protein function which is the current passing through the channel.
In addition, the advancements in cryoEM allow potentially for the capturing of multiple conformations of an ion channel. We are currently using this approach to try and determine multiple states of ASICs in a real bilayer.