Fluorescence anisotropy decay demonstrates calcium-dependent shape changes in photo-cross-linked calmodulin.
We report dynamic fluorescence anisotropy measurements on the purified dityrosine derivative of calmodulin which was generated during UV irradiation of Ca2+-containing solutions of bovine brain calmodulin [Malencik, D. A., & Anderson, S. R. (1987) Biochemistry 26, 695]. Measurements were made by using a high repetition rate picosecond laser source combined with a microchannel plate photomultiplier. This permits the collection of very low noise anisotropy curves with essentially no convolution artifact. Measured anisotropies at high calcium concentrations are monoexponential, and at 20 degrees C, we recover a correlation time of 9.9 ns. When the temperature is varied from 4.8 to 31.8 degrees C, the recovered correlation time is proportional to the viscosity and inversely proportional to the absolute temperature, behavior expected for the rotational diffusion of a macromolecule whose conformation is independent of the temperature. The correlation time is compared to the theory describing the rotational diffusion of a dumbell. At high calcium concentrations, the cross-linked calmodulin is elongated and has a length equal or nearly equal to that predicted by X-ray crystallographic results. In the absence of calcium, the molecule becomes highly compact and exhibits significant segmental motion. Intermediate calcium ion concentrations result in an intermediate degree of elongation and segmental motion. A small increase in the measured rotational correlation time of calmodulin upon the binding of melittin and mastoparan indicates that these peptides cause no major changes in the elongation of the molecule. When the cross-linked calmodulin is bound to troponin I, the complex rotates as a unit with a single rotational correlation time of 22 ns.
Small
E W
EW
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331.
Anderson
S R
SR
D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D005454:Fluorescence Polarization; D007700:Kinetics; D008433:Mathematics; D008958:Models, Molecular; D009025:Morpholines / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D014961:X-Ray Diffraction
D002147:Calmodulin; D003432:Cross-Linking Reagents; D009025:Morpholines; C008550:morpholinopropane sulfonic acid; D002118:Calcium