Project Summary In this proposal we seek to develop single molecule FRET in prestin. The method will allow us to assess the function of the protein using optical measures. Single molecule FRET has been developed in other proteins to aid in the understanding of their function. Presently, we do not have a functional assay of prestin with a single molecule resolution. The rationale for the experiments is based on structural data interpolated from the structure of several members of the extended anion transporter family to which prestin belongs. Available structural data together with modeling data suggest that members of this family have 14 transmembrane domains with an inverted repeat 7+7 structure to each monomer. The functional units of these proteins appear to be dimers. Each monomer has a gate and core domain. We model prestin to behave like these transporters using an elevator mechanism to move from contracted to expanded states. An analogous movement in these family members from inside open to outside open conformations result in a relative motion between two parts of the same monomer. Thus, there is a movement of the gate domain (Tm domains 5-8 and 12-14) relative to the stable core domain (Tm domains 1-4 and 9-11) containing the Tm anion binding site. Based on these models we will insert several cysteine residues in the gate domain that will allow us to label a single dimer with two different fluorophores with FRET activity. We have already generated a cysteine free protein that shows reduced but measurable gating charge movement (NLC), confirming its functionality. The distance between the fluorophores is modelled to vary by 30A0 that would give rise to a significant change in FRET efficiency. Establishing a single molecule FRET assay will allow us to measure how physiological parameters affect the function of the protein.