FLUORESCENCE ENERGY-TRANSFER BETWEEN METAL-IONS IN THERMOLYSIN - THERMAL-DENATURATION STUDIES

Thermolysin derivatives have been prepared with one zinc ion, two calcium ions, and one terbium ion. Excitation of this derivative at 280 nm results in emission of visible Tb3+ fluorescence at 545 nm. When Co2+ is substituted for the Zn2+, the Tb3+ emission is quenched due to energy transfer between the Co2+ and Tb3+. Distances have been calculated between the two metal ion binding sites assuming a dipole-dipole mechanism for energy transfer (Berner, V. G., et al. (1975) Biochem. Biophys. Res. Commun. 66, 763; Horrocks, W. D., Jr., et al. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4764). We have extended these studies by following the Co2+-Tb3+ distances as a function of temperature. We have found a gradual increase in the distance between the two metal ions that ranges from nearly 14 A at 25 °C to nearly 22 A at temperatures above 80 °C. Conventional techniques for measuring conformational changes in proteins (tryptophan-tyrosine fluorescence, optical rotation or enzyme activities) show little or no change in the protein structure up to 70 °C. At higher temperature drastic changes in thermolysin properties indicate an extensive unfolding of the protein. In contrast to the optical rotation, fluorescence, and activity measurements, viscosity measurements indicate changes in the protein structure at temperatures well below 70 °C, and these changes correspond rather well with the distance changes between the Co2+ and Tb3+. This indicates that both the viscosity and fluorescence energy transfer experiments are detecting changes in the protein structure which are not detectable by the other techniques. © 1978, American Chemical Society. All rights reserved.