Decomposing NMR Ensemble with the Assistance of Single Molecule FRET

Abstract

Proteins are inherently dynamic, and the dynamics allow the protein to fulfill specific functions. NMR is exquisitely sensitive to protein dynamics. In particular, paramagnetic relaxation enhancement (PRE) has an <r−6> distance dependence, and has been used to visualize sparsely populated protein conformation(s). However, owing to the averaging of NMR signals over many different copies of protein molecules, it can be difficult to resolve the constituting conformational states that contribute to the observables. Single molecule techniques, on the other hand, can be used to visualize the fluctuation of a single protein molecule over time. Here I will present how the characterization of protein ensemble structures can be made easier and more accurate with the incorporation of single molecule FRET data. The relative populations of the inter-converting conformational states can be obtained from single-molecule measurement, and detailed structural information can be characterized by NMR and other biophysical techniques. Moreover, the calculated ensemble structures of the protein can be verified by the distance information from single molecule FRET. Using both bulk and single-molecule techniques, we have resolved and visualized the ensemble structures of polyubiquitin and other multi-domain/subunit proteins. We envision that such an integrative approach will play an increasingly important role in characterizing the dynamics of biological macromolecules.