BIOLOGICAL CONCENTRATION REPORTERS:
Exploring a Dual GFP Construct at the Single Molecule Level

Figure 1: Schematic of the "Cameleon YC 2.1" construct and the principle of calcium concentration detection by FRET described in the text.

(Brasselet, Peterman, Miyawaki, and Moerner, J. Phys. Chem. B (published on the web, March 2000), also J. Phys. Chem. B 104, 3676-3682 (2000))

One thrust of our research concerns the photophysical behavior of single molecules of the Green Fluorescent Protein (GFP) and its mutants. As is well-known, in cell biology and biochemistry, GFP is currently widely used as an indicator for gene expression or as a fluorescent label for a large variety of proteins. In previous work, we observed single copies of GFP for the first time (Dickson, Cubitt, Tsien, and Moerner, Nature, 355, 388 (1997)), and our paper characterizing the fascinating single-molecule blinking dynamics as a function of pH, host, mutant, and excitation intensity has recently appeared (Peterman, Brasselet, and Moerner, J. Phys. Chem. A 103, 10553 (1999)).

As a critical extension of this work, we have explored the single-molecule dynamics of dual-GFP constructs designed to sense local ion concentrations in biological media. A first study, recently completed, concerns the "cameleon YC2.1" complex, whose structure is based on a cyan-emitting GFP (CFP) separated from a yellow-emitting GFP (YFP) by the calmodulin Ca2+-binding protein (CaM) and a calmodulin-binding peptide (M13) (see Fig 1). This complex was provided by R. Y. Tsien, and it was designed to allow sensing of calcium ion concentrations in cells by fluorescence resonant energy transfer (FRET). If Ca2+ ions are bound, CaM wraps around M13, and the construct forms a more compact shape, leading to a higher efficiency of excitation transfer from the donor CFP to the acceptor YFP. The degree of FRET in cameleon is therefore a sensitive ratiometric reporter of the concentration of Ca2+ in solution and cells. We measure FRET at the single-molecule level using an ultrasensitive two-color confocal scanning microscope.

Analysis of single-molecule signals from the cameleon YC2.1 complex diluted in aqueous agarose gels allowed retrieval of several interesting features of the energy transfer between the donor and acceptor mutants of the construct, as a function of the calcium concentration in the medium. The energy transfer efficiency distribution deduced from single-molecule fluorescence signals shows an increased width at the Ca²⁺ dissociation constant concentration (see Figure 2). This observation is consistent with the ligand binding kinetics, whose time scale at intermediate calcium concentration is close to our measurement time scale (20 ms). The complex dynamics of the fluctuations were examined using a combination of autocorrelation and cross-correlation in conjunction with polarization measurements at the bulk and single-molecule level. Beside the reorientation fluctuations of the two dipoles that seem to occur on a time scale fast compared to our integration time (<20ms) but slow compared to the fluorescence lifetime, we detected variations in the energy transfer between the two GFP mutants on the 20-100 ms time scale. We found both negative and positive cross-correlations in the donor and acceptor emission signals, the former related to the energy transfer process, and the latter presumably caused by other perturbations of the donor and acceptor emission. A further conclusion of this work is that single copies of this dual-GFP construct cannot accurately report local calcium concentrations, chiefly due to the small number of photons emitted before the termination of the on-time. Future studies should explore methods to terminate the dark period by secondary optical excitation.

Figure 2: Histograms of the energy transfer efficiency of cameleon YC2.1 in agarose deduced from single-copy measurements of for three different concentrations of calcium:  at Ca2+ saturation, at an intermediate concentration (~ Kd), and at very low calcium.