Interchange of aequorin and obelin bioluminescence color is determined by substitution of one active site residue of each photoprotein

The bioluminescence spectra from the Ca2+-regulated photoproteins aequorin (λmax=469nm) and obelin (λmax=482nm) differ because aequorin has an H-bond from its Tyr82 to the bound coelenteramide, not present in obelin at the corresponding Phe88. Substitutions of this Phe88 by Tyr, Trp, or His shifted the obelin bioluminescence to shorter wavelength with F88Y having λmax=453nm. Removal of the H-bond by the substitution of Y82F in aequorin shifted its bioluminescence to λmax=501nm. All mutants were stable with good activity and were expressible in mammalian cells, thereby demonstrating potential for monitoring multiple events in cells using multi-color detection.