Nanosecond fluorescence dynamic Stokes shift of tryptophan in a protein matrix

The fluorescent dynamic Stokes shift (FDSS) method has emphasized a time-dependent dipolar relaxation process around the single tryptophan residue (Trp31) in cytidine monophosphate kinase from E. coli (CMPK). This Trp residue, located close to the protein surface in a hydrophobic pocket, is weakly accessible to acrylamide, a water-soluble quencher. It exhibits fluorescence characteristics suitable for a detailed study of dipolar relaxation: (i) a fluorescence decay almost monoexponential and (ii) a fluorescence emission maximum of 329 nm, in a wavelength range intermediate between those of a completely polar environment and a strongly apolar one. This emission maximum is shifted to 320 nm by decreasing the temperature to 230-240 K with glycerol as cryoprotectant. A time constant (similar to 100 ps) affected by a negative preexponential, evidenced in the red-edge fluorescence intensity decays, supports the existence of an excited-state reaction. A multiphasic FDSS (with time constants ranging from similar to 100 ps to several nanoseconds) with a total amplitude between 130 and 340 cm(-1) (0.4-1 kcal.mol(-1)) is observed in the temperature range 293-232 K and not below.