Cameleons
Fluorescent probes such as Fura-2, Indo-1, and Fluo-3 are popularly used for measuring calcium ion concentration in cells. In 1997, Dr. Atsushi Miyawaki (Riken Brain Science Institute, Wako, Japan) developed a novel probe for calcium ion measurement. The probe, an artificial protein modified from green fluorescent protein (GFP) (Nature, 388: 882-887, 1997), was named Cameleon after the animal. Cameleon consists of two fluorescent proteins, calmodulin, and M13. Calmodulin can bind with calcium ions and M13 can bind with calmodulin bound with calcium ions. The genes of these four proteins are joined linearly, and the fusion genes are expressed in a variety of cells.
Cameleon is remarkable in its practical applications in genetic engineering and Fluorescence Resonance Energy Transfer (FRET), a biophysics technique for measuring the distance between two molecules. A pair of fluorescent proteins for FRET is selected according to their characteristics of absorption and emission spectra. The excitation of one fluorophore (the donor) is transferred to another (the acceptor) without fluorescence emission in which donor emission and acceptor absorption overlap spectrally. Energy transfer takes place only when the two fluorophores are closed. A CFP/YFP pair and a BFP/GFP pair are generally chosen because of their spectral characteristics in fluorescent protein variants (see CLONTECH website).
The Cameleon molecule consists of four domains (See below.). Fluorescent proteins joined at both ends stand away from each other in the absence of calcium ion. In the presence of calcium ion, the activated CaM wraps around the M13 protein. The structure of Cameleon then changes to approximate the distance between CFP and YFP. In a low calcium concentration environment, many Cameleon molecules take an unfolded structure and CFP fluorescence is clearly. When the calcium ion concentration increases, CFP fluorescence decreases and YFP fluorescence increases due to Fluorescence Resonance Energy Transfer (FRET).
Fig.1
Intracellular calcium ion concentration can be determined by fluorescence ratio imaging as the ratio of the changes in two types of Cameleon molecules according to the intracellular concentration of calcium ion. When Cameleon has been excited at the 442 nm line of an He-Cd laser, its fluorescence has been detected simultaneously at 485 nm and 530 nm on confocal laser scanning microscopy.
Fig.2
In the above experiment, we used the split type of Cameleon that consists of two fusion genes, CFP-CaM and M13-YFP. The two genes were expressed simultaneously in HeLa cells by Lipofectamine (GIBCO; Invitrogen, Carlsbad. CA, USA). Stimulation by 0.1 mM of Histamine caused the intracellular concentration of calcium ion in HeLa cells to increase. This increase was inhibited by 0.1 mM of Cyproheptadine with antagonistic actions.
