MUTANT FLUORESCENT PROTEINS 
Spectral GFP mutants were made, which allow tracking of several fusion proteins in one cell simultaneous. So in addition to species related optimization also GFPs with markedly modified fluorescence properties have been made. This resulted in FPs with shifted emission spectra, such as Blue FPs, Yellow FPs, Red FPs and Purple FPs. Beside these GFP derivatives new GFPs with different spectral properties were discovered for example Discosoma sp. DsRED and Heteractis crispa HcRED, All together this resulted in a good collection of FPs with different excitation and emission spectra.
FIG 1: Normalized excitation (a) and emission (b) spectra of several fluorescent proteins. Enhanced Blue FP(EBFP), Cyan FP (ECFP), Green FP (EGFP), Yellow FP (EYFP), Red FPs (DsRed from Discosoma sp. and it monomeric RFP (mRFP1) derivative and HcRed1 from Heteractis crispa
Siemering K. R., Golbik R., et alt., Curr. Biol., 1996, 6, 1653–1663
As shown the excitation and emission spectra of the FPs are sufficiently different to select FP couples that can be tracked simultaneously. It turned out even to be possible to follow 4 FPs simultaneously. Additional spectral data off FPs and other fluorescent dyes can be found at Pubspectra. Mutations in GFP not only resulted in spectral variants, but also in some cases the spectral properties of mutated GFPs became depended on environmental parameters like intracellular ion concentrations or redox potentials. Such mutated forms can be used as sensors and provide subcellular information about such. In addition to these more or less coincidently obtained sensor GFPs, sensors also can be tailor made by inserting a specific protein domain into the cylindrical s-can structure of GFP. An example of such a domain is the calmoduline domain, which can bind the cellular compound, calcium (Ca2+). Upon calcium binding the calmoduline-GFP barrel structure is distorted upon which also the fluorescence emission is altered.
FIG 2: Examples of mutant proteins
The currently known GFP variants may be divided into seven classes based on the distinctive component of their chromophores:
WILD-TYPE MIXTURE OF NEUTRAL PHENOL AND ANIONIC PHENOLATE (CLASS 1)
PHENOLATE ANION IN CHROMOPHORE (CLASS 2)
NEUTRAL PHENOL IN CHROMOPHORE (CLASS 3)
PHENOLATE ANION WITH STACKED p -ELECTRON SYSTEM (CLASS 4)
INDOLE IN CHROMOPHORE DERIVED FROM Y66W (CLASS 5)
IMIDAZOLE IN CHROMOPHORE DERIVED FROM Y66H (CLASS 6)
PHENYL IN CHROMOPHORE DERIVED FROM Y66F (CLASS 7)
We analyzed these mutant proteins: