Thermal profiles of förster energy transfer preliminary studies of luminescent probes of protein dynamics in transferrin and calmodulin

Fluorescence energy transfer, the transfer of energy from a donor to an acceptor via a dipole/induced dipole mechanism, has long been used to measure distances between donors and acceptors in proteins and other macromolecules. Because the transfer can occur over time scales larger than protein bending and breathing modes, multiple conformational states can be sampled. The analysis of these states is weighted by the donor-acceptor distance; shorter distances carry more weight, because the energy transfer depends on the inverse sixth power of the distance. The usefulness of fluorecence energy transfer in probing these large amplitude protein motions is studied here. The method involves measuring the nergy transfer efficiency while perturbing the protein conformation 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 flexible region of the protein. This hypothesis was tested in two different protein systems; calmodulin, a calcium- activated regulatory protein, and transferrin, a blood serum iron shuttle. The preliminary studies show a differential sensitivity of the transfer efficiency to heat for the systems. Normalized energy transfer over 10 Å in calmodiulin from a tyrosine donor to a Tb(III) acceptor increases 40% from 297 to 322 K. Normalized energy transfer over 42 Å in transferrin from a Tb(III) donor to an Fe(III) acceptor increase 35% over the same temperature range. In marked contrast to these systems, energy transfer from tyrosine to a chelated Tb(III) shows anomalously high temperature- dependence.