title: Fluorescence studies of phosphoribulokinase, a light-regulated, chloroplastic enzyme.
abstract: Recent characterization of spinach phosphoribulokinase has revealed that the homodimeric molecule contains only two tryptophans per 44-kDa subunit. We have performed steady-state and frequency domain studies of the intrinsic fluorescence of this protein. The fluorescence properties reflect contributions from both types of tryptophan residues. One of these appears to be relatively exposed to solvent and the quencher, acrylamide; fluoresce with a lambda max of 345 nm; decay with a fluorescence lifetime of 6.3 ns; have a relatively red-shifted absorption spectrum; and have a certain degree of independent motional freedom, with respect to the protein. The other tryptophan residue appears to be more buried; fluoresce with lambda max of 325 nm; have a lifetime of 1.7 ns; have a relatively blue-shifted absorption spectrum; and not to enjoy independent motional freedom. On comparison of phase-resolved spectral data and solute quenching data, we suggest that resonance energy transfer between the blue and red tryptophan residues may occur. We also describe the strategy of simultaneously fitting Stern-Volmer quenching data collected at two emission wavelengths.
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia 65201.
authors.forename: C A
authors.initials: CA
authors.lastname: Ghiron
authors.forename: M R
authors.initials: MR
authors.lastname: Eftink
authors.forename: M A
authors.initials: MA
authors.lastname: Porter
authors.forename: F C
authors.initials: FC
authors.lastname: Hartman
mesh_terms: D002736:Chloroplasts / Q000201:enzymology*; D008027:Light; D010770:Phosphotransferases / Q000032:analysis; D017853:Phosphotransferases (Alcohol Group Acceptor); D010945:Plants, Edible / Q000201:enzymology; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors; D014364:Tryptophan / Q000032:analysis
chemical_list: D014364:Tryptophan; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase