Single-Molecule Resonance Energy Transfer and Fluorescence Correlation Spectroscopy of Calmodulin in Solution

Calmodulin is a calcium-signaling protein that is involved in a diverse range of biological pathways. The flexibility of the two lobes of CaM about a central linker domain is crucial to target recognition and binding. We have attached fluorescent probes to the N-terminal and C-terminal domains of CaM. In this study, we report single-molecule Förster resonance energy transfer (FRET) between the two domains. We have detected fluctuations in single-molecule FRET efficiency on the microsecond and millisecond time scales by fluorescence correlation spectroscopy (FCS). The cross-correlation decay due to FRET on the 100 μs time scale is sensitive to the Ca 2+ concentration, with similar relaxation at a saturating Ca 2+ concentration of 100 μM and in the absence of Ca 2+, but distinctly slower relaxation in the presence of 1 μM Ca 2+ . We have also measured the FRET efficiency distribution by analysis of fluorescence bursts in solution. The distributions of single-molecule FRET efficiencies reveal the existence of multiple conformations in solution. At least two distinct conformations are detected and attributed to distinct configurations of the N- and C-terminal domains about the central linker of CaM. These distributions are confirmed by time-resolved ensemble FRET measurements. In addition, FCS yields the diffusion coefficient for CaM. We discuss in detail issues involved in analysis of single-molecule FRET measurements, including analysis of signals and the nature and effect of dye interactions with the protein.