CAMELEON

Since the first report of cameleon, sensors based on the same approach have been designed to detect small molecules (including cAMP [162, 163], cGMP [164], Ins(l,4,5)P3 [165, 166], lipids [167, 168], and sugars [169]) and the activity of a wide variety of kinases [122, 168, 170, 171]. 

Evanko, D. S. and Haydon, P. G. (2005). Elimination of environmental sensitivity in a cameleon FRET-based calcium sensor via replacement of the acceptor with Venus. Cell Calcium 37, 341-8. 

Several other approaches to solve the quantitation problem have been proposed. Hoppe et al. [2] determined y/ by calibrating it against constructs with known FRET efficiency. We and others [3, 6] have used data from a cell before and after acceptor photobleaching to relate the FRET-induced sensitized emission in the S channel to the loss of donor emission in the D channel by factors termed or G, respectively. For the CFP/YFP pair this works very well on confocal microscopes with a 514-nm Argon ion laser line, but on wide-held systems, selective acceptor photobleaching reportedly causes problems [ 14]. F inally, G can also be determined by comparison of several constructs that differ in FRET efficiency, a bit analogous to the Yellow Cameleon calibration described above [10,14],... 

Sensor any single-polypeptide construct (containing the fluorophores to be used in the experiment) that can be induced to change FRET significantly and homogeneously e.g., Yellow Cameleon in combination with ionomycin. 

Miyawaki, A., Griesbeck, O., Heim, R. and Tsien, R. Y. (1999). Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc. Natl. Acad. Sci. USA 96, 2135-40. 

Demaurex, N. and Frieden, M. (2003). Measurements of the free luminal ER Ca(2+) concentration with targeted cameleon fluorescent proteins. Cell Calcium 34, 109 19. 

Chevillot and Edwards, Mdmoire sur le cameleon mineral, Ibid, (2),... 

Fiala A., Spall T., Diegelmann S., Eisermann B., Sachse S., Devaud J.-M., Buchner E. and Galizia C.-G. (2002) Genetically expressed cameleon in Drosophila melanogaster is used to visualize olfactory information in projection neurons. Curr. Biol. 12, 1877-1884. 

Cameleon - single-pair FRET between two GFP mutants in a [95]... 

As is well-known, FRET is a powerful tool for sensing conformational changes in single biomolecules [92-94]. The first FRET study of a single dual-GFP construct was completed in the Moerner lab for the cameleon YC2.1 complex in 2000 [95]. Analysis of single-molecule signals from the... 

Fig. 2.10. (A) Schematic of the structure of cameleon containing CFP, YFP, CaM, and M13 showing the change in FRET upon binding and unbinding of Ca + ions. (B) Histograms of the energy transfer efficiency measured for single molecules of cameleon in agarose gels, at three different Ca ion concentrations. For details, see [95]...

Figure 2. Improvement of yellow cameleon by using a circularly permuted YFP variant. (A) The three-dimensional structure of GFP with the positions of the original (Metl) and new N-termini (Aspl73). (B) Domain structures of YCS.12 and YC3.60. CaM, Xenopus calmodulin Ml3, a CaM binding peptide derived from myosin light chain kinase (C, D) Emission spectra of YCS. 12 (C) and YC3.60 (D) (excitation at 435 nm) at zero (doted line) and saturated Ca (solid line). (E) A series of confocal pseudo-B/W images showing propagation of [Ca c- These images were taken at...

Constructs 3D structures from SMILES notation using a library of fragments and rules. ASP for molecular similarity comparisons, CONSTRICTOR for distance geometry, and CAMELEON for protein sequence alignment. NEMESIS for desktop molecular modeling on the Mac-II and IBM PC. 

S. Habuchi, M. Cotlet, J. Hofkens, G. Disix, J. Michiels, J. Vanderleyden, V. Subramaniam, F.C. De Schryver, Resonance Energy Transfer in a Calcium Concentration-Dependent Cameleon Protein, Biophys. J. 83, 3499-3506 (2002)... 

FRET imaging can monitor dynamic interactions between a FRET donor (ECFP) and an acceptor (EYFP), which are fused to the protein of interest, in live cell experiments. As an example, we show the FRET changes of a fluorescent biosensor for Ca +, came-leon, using the spectral detector of the Zeiss LSM 510 META in a time-lapse imaging experiment. Cameleon is a chimeric protein that consists of ECFP, the calmodulin-binding peptide M13... 

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