Site-specific Incorporation of 19F into Arrestin-3 as an NMR Probe for Characterizing Its Conformational Changes

  • Rebecca Stout


"Signal specificity is achieved through highly-regulated molecular recognition. Arrestins, a small family of multi-functional adaptor proteins, mediate surprisingly complex cellular signaling events. To elicit the appropriate cellular response, arrestins have to ensure correct components are linked together to prevent the formation of mismatched protein complexes. It is now clear that arrestins undergo significant conformational movements in many regulating processes; however, previous studies of arrestin conformational changes are almost completely focused on receptor-induced conformational changes, and little is known about binding-induced conformations that allow arrestins to accomplish their diverse signaling functions. We hypothesize that binding-induced conformational changes play decisive roles in regulating the recruitment of binding partners.
19F NMR has proven to be a powerful technique in the study of protein structure and dynamics because the 19F nucleus is easily incorporated into protein through specific labeling. The chemical shift of 19F is extremely sensitive to changes in the local microenvironment caused by the conformational movements of protein, making it possible to analyze minor movements within a protein molecule. 19F NMR is performed by incorporating a fluorine label into the protein structure Fluorine labeling can be accomplished in a variety of ways. Here, we developed a 19F NMR-based method to detect conformational movements of arrestins by employing a thiol-based method to incorporate 19F to specific arrestin residues. We have observed the release of the C-tail in arrestin-3 upon binding of inositol hexaphosphate (IP6). The methods developed here can be employed to detect the conformational changes of arrestin with many other binding partners."