17] The use of singlet-singlet energy transfer to study macromolecular assemblies

Abstract

Publisher Summary Fluorescence spectroscopy has assumed an increasingly active role in the study of the dynamics and structure of biological macromolecular assemblies. The most definitive information available from fluorescence measurements is the distance between the pairs of loci in the assembled structure. The distance is determined from the efficiency of nonradiative singlet-singlet energy transfer between the pairs of fluorescent dyes. The rate of this transfer is proportional to the inverse sixth power of the distance between a given pair of chromophores. By appropriate choice and placement of fluorescent dyes, a distance that corresponds either to the specific distance between two chemically unique points in the assembled structure, or to the center-to-center distance between two globular components in the structure can be determined. The basic approach described in this chapter is to label one region of the assembly with a fluorescent energy donor, label a second region with an energy acceptor, measure the efficiency of nonradiative energy transfer between the donor and acceptor, and then convert this efficiency to a distance between the two labeled regions. The result of these studies has shown that singlet–singlet energy transfer has the ability to generate structural details of complex macromolecules in solution. While this technique has its limitations and is often tedious because distances must be measured one at a time, singlet–singlet energy transfer offers one major advantage over other techniques that have been applied to the study of macromolecular assemblies; unlike X - ray diffraction neutron scattering, electron microscopy, and cross-linking, fluorescent techniques can be carried out on dilute solutions of assemblies that maintain full biological activity.