Fluorescence characterization of VU-9 calmodulin, an engineered calmodulin with one tryptophan in calcium binding domain III.
Absorption and fluorescence properties of VU-9 calmodulin, an engineered calmodulin in which a tryptophan residue has been introduced in position 99, have been investigated. Tryptophan 99 fluoresces with a maximum around 348 nm and is easily quenched by fluorescence quenchers such as acrylamide, indicating that the chromophore is in a polar environment and well exposed to the solvent, a location which has been reported previously for tyrosine 99 in mammalian calmodulin [Kilhoffer, M. C., Demaille, J. G., & Gérard, D. (1981) Biochemistry 20, 4407-4414]. The quantum yields of tryptophan 99 were found to be 0.19 in the absence of calcium and 0.15 in its presence. These values indicate that the chromophore is in a particular microenvironment where it is protected from the quenching mechanisms normally occurring in proteins. Steady-state fluorescence polarization measurements indicate that the protein exhibits segmental mobility both in the absence and in the presence of calcium. Binding of calcium decreases the mobility of the chromophore, a good indication for a rigidification of the protein structure. A quite rigid structure of at least the carboxy-terminal part of VU-9 calmodulin in the presence of Ca2+ is also suggested by Förster energy-transfer measurements.
Laboratoire de Biophysique, Faculté de Pharmacie de Strasbourg, UA CNRS 491, Université Louis Pasteur, Illkirch, France.
M C
MC
Kilhoffer
D M
DM
Roberts
A
A
Adibi
D M
DM
Watterson
J
J
Haiech
D001665:Binding Sites; D002118:Calcium; D002147:Calmodulin; D004735:Energy Transfer; D005454:Fluorescence Polarization; D011789:Quantum Theory; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D002147:Calmodulin; D011994:Recombinant Proteins; D014364:Tryptophan; D002118:Calcium