Fluorescence Probes of Protein Dynamics and Conformations in Freely Diffusing Molecules

The results described here demonstrate that single-molecule fluorescence spectroscopy of molecules freely diffusing in solution can yield unique information about both the dynamics and conformations of proteins. We used single-molecule FRET to detect the dynamics in CaM on the timescales of 100’s of microseconds and a few milliseconds by cross-correlation analysis of the donor and acceptor signals. In addition, analysis of the distribution of donor-acceptor distances by single-molecule FRET demonstrates the presence of three distinct conformational substates of CaM in solution. Together these results paint a picture of a protein that is dynamic and flexible, accessing a range of distinct conformation substates in solution.