Single-molecule fluorescence resonant energy transfer in calcium concentration dependent cameleon

The degree of fluorescence resonant energy transfer in the protein construct "cameleon", whose structure is based on two green fluorescent protein mutants and the calmodulin Ca2+-binding protein, is a sensitive ratiometric reporter of the concentration of Ca2+ in solution and cells. For the first time, the detection of single copies of cameleon immobilized in agarose gels is described. This work exemplifies the issues involved in the observation of binding kinetics at the single-molecule level, using fluorescence resonant energy transfer as a signature of the response of the system to the presence of Ca2+ ions. The energy transfer distribution deduced from single-molecule fluorescence signals shows an increased width at the Ca2+ dissociation constant concentration, which is consistent with the ligand binding kinetics. The complex dynamics of the fluctuations are examined using a combination of autocorrelation and cross-correlation in conjunction with polarization information. We find both negative and positive cross-correlations in the donor and acceptor emission signals, the former related to the energy transfer process, and the latter caused by other perturbations of the donor and acceptor emission.