Abstract
We have initiated an investigation into the usefulness of fluorescence energy transfer in probing protein dynamics. Our analysis involves measuring the energy transfer efficiency while perturbing the protein conformational equilibrium with heat. As the temperature increases, the amplitudes of vibrations increase, and fluorescence energy transfer should also increase if the donor and acceptor are in a flexible region of the protein. A theoretical analysis developed by Somogyi and co-workers for the temperature dependence of dipole-dipole energy transfer (Somogyi, B., J. Matko, S. Papp, J. Hevessey, G. R. Welch, and S. Damjanovich. 1984. Biochemistry. 23:3403-3411) was tested by the authors in one protein system. Energy transfer from tryptophan to a pyridoxamine derivatized side group in RNase increased 40% over 25 degrees C. Here we report further testing of this model in two additional protein systems: calmodulin, a calcium activated regulatory protein, and transferrin, a blood serum iron shuttle. Our studies show a slight differential sensitivity of the transfer efficiency to heat for the two systems. Normalized energy transfer over 6.5 A in calmodulin from a tyrosine donor to a Tb(III) acceptor increases 40% from 295 to 320 K. Normalized energy transfer over 42 A in transferrin from a Tb(III) donor to an Fe(III) acceptor increases 35% over the same temperature range. Whereas these results demonstrate that thermally induced fluctuations do increase energy transfer as predicted by Somogyi, they also appear rather insensitive to the nature of the protein host environment. In contrast to the Förster processes examined above, energy transfer over very short distances has shown an anomalously high temperature dependence.