Small and rapid molecular motions often drive dramatic changes in protein systems. Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique to probe conformational dynamics at the nanometer scale. In this aim, we plan to improve the spatial and temporal resolution of smFRET to probe the motions of proteins, revealing the interplay between nanoscale motion and the macroscopic function. We also access these length scales in a near physiological environment while accurately monitoring the dynamics of the structural changes. These two improvements enable a new, mechanistic understanding of ligand binding to receptors and other nanoscale molecular motions.
Single molecule FRET assay
Overall, nanoscale spatial and microsecond temporal resolution is required to differentiate the response of receptor proteins to ligand binding events. Indeed, the approach presented here provides a new mechanistic basis to optimize a wide array of human therapeutics, and to understand the nanoscale motions that underlie biological function.
The rate of energy transfer between two molecules is quantified as a function of the distance between them [1]. Subsequently, it was shown that this relationship could be exploited to provide a 'spectroscopic ruler' by measuring the efficiency of energy transfer because the efficiency increases with the rate [2]. This advancement led to determination of inter- and intraprotein distances, offering new insight into protein structure, conformational dynamics, and interaction [3] within single molecule resolution [4]. In particular, single-molecule FRET has provided new insight into conformational dynamics, because these processes are asynchronous, and thus average out in ensemble approaches.
In studying conformational dynamics, FRET has two key advantages over other structural techniques: (1) Measurements can be performed in near-physiological environments; and (2) Nanometric structural dynamics can be probed providing the ability to monitor the timescales and sizes of structural motions. While ensemble FRET can be adapted to resolve distances down to ~1 nm, smFRET is much more difficult to extend below 3 nm because of low signal to background at these distances. Here, we extend the scope of smFRET to obtain resolution of 1nm by directly measuring the rate of energy transfer, instead of the efficiency, using existing smFRET fluorophore technology.