Time-resolved fluorescence study of VU-9 calmodulin, an engineered calmodulin possessing a single tryptophan residue.
An engineered calmodulin (VU-9 calmodulin), which possesses a single tryptophan residue at position 99 in calcium binding domain III, was studied by time-resolved fluorescence. At least two exponential terms are needed to describe the tryptophan fluorescence decays, either in the presence or in the absence of calcium. The characteristics of the fluorescence decays are strongly dependent upon the number of calcium ions bound per molecule of VU-9 calmodulin until half of the calcium sites are occupied, i.e., three in the absence of magnesium and two in the presence of 5 mM magnesium. A clear time-dependent spectral shift is observed in the presence of calcium. The existence of an isosbestic point in the time-resolved spectra is in agreement with a two-state model. The biexponential analysis of the 340-nm fluorescence decay during calcium titration gives parameters consistent with a two-state model in which tryptophan 99 interconverts between two different conformations, characterized by a different lifetime value, with rates altered by calcium binding. This model explains the decrease in the protein quantum yield induced by calcium binding [Kilhoffer, M. C., Roberts, D. M. Adibi, A. O., Watterson, D. M., & Haiech, J. (1989) Biochemistry (preceding paper in this issue)].
Chabbert
M
M
Laboratoire de Biophysique, Faculté de Pharmacie de Strasburg, UA CNRS 491, Université Louis Pasteur, Illkirch, France.
Kilhoffer
M C
MC
Watterson
D M
DM
Haiech
J
J
Lami
H
H
D001665:Binding Sites; D002118:Calcium; D002147:Calmodulin; D007700:Kinetics; D011487:Protein Conformation; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D002147:Calmodulin; D011994:Recombinant Proteins; D014364:Tryptophan; D002118:Calcium