Single-Molecule Measurement of Membrane Protein Stability

Our understanding of membrane proteins has been severely hindered by a dearth of techniques to investigate membrane protein folding and stability in lipid bilayers. Our lab has developed a technique to drive membrane protein unfolding under native conditions called the steric trap method, which uses a protein that preferentially binds the unfolded state such that binding drives unfolding. Previously, this method has been reliant on the use of a functionally assayable protein, such as the chromophore-containing protein bacteriorhodopsin or the membrane enzyme diacylglycerol kinase. We propose generalizing the steric trap method using single-molecule fluorescence technology. By moving the system to infinite dilution in which there is only a single protein per vesicle we can eliminate aggregation problems that plague membrane protein studies. The sensitivity of single-molecule detection permits us to use much less protein and lipid material, and the minimal light scattering allows us to observe proteins in vesicles instead of using solubilizing agents required for bulk spectroscopic assays. By circumventing the requirement for a protein-specific functional assay, we hope to expand the reach of membrane protein folding studies.