Using structure-function constraints in FRET studies of large macromolecular complexes

The structural aspects of large macromolecular systems in solution can be conveniently addressed using the fluorescence resonance energy transfer (FRET) approach. FRET efficiency is the major parameter examined in such studies. However, its quantitative determination in associating macromolecular systems requires careful incorporation of thermodynamic quantities into specific expressions defining the FRET efficiencies. There are two widely used methods of obtaining FRET efficiencies, examination of both the donor quenching and of the sensitized emission of the FRET acceptor. Both approaches provide only apparent FRET efficiencies, not the true Förster FRET efficiency, which should be independent of the means to measure the efficiency. The accuracy of the determined distances in macromolecular systems depends on the accuracy of the determination of the FRET efficiency and the estimate of the parameter, κ ², which depends on the mutual orientation of the donor and the acceptor. Known procedures, based on limiting anisotropy measurements, to estimate κ ² are of limited use to deducing the functional conclusions about the studied systems. On the other hand, using multiple donor-acceptor pairs and/or donors and acceptors placed in interchanged locations in the macromolecular system is an equally rigorous procedure to empirically evaluate the possible effect of κ ² on the measured distance. Protein-nucleic acid systems are particularly suited for FRET methodology. There is a plethora of commercial fluorescent markers, which can serve as donor-acceptor pairs. In the case of the nucleic acid, the markers can specifically be introduced in practically any location of the molecule. Application of the FRET measurements to examine structures of the large protein-nucleic acid complexes is particularly fruitful in cases where the presence of known structural constraints allows the experimenter to address the fundamental topology of the complexes. The discussed methodology can be applied to any associating macromolecular system.