Fluorescence quenching in proteins: some applications to protein-DNA and protein-lipid interactions

摘要

Fluorescence quenching is a powerful technique for assessing local structure of macromolecules and their assemblies. In addition to determination of "accessibility" (via collisional quenching) and "affinity" (for complexes that yield static quenching), quenching due to resonance energy transfer (RET) is widely used to ascertain distances between probes. In this paper, we will outline some examples of the advantages found in subdividing overall quenching into heterogeneous contributions. Subdivision is accomplished by overdetermination (global) and association (DAS, decay associated spectral) methods. In some cases, the subdivision of fluorescence leads to unique identification of different fluorophores in different sites. Alternatively, the recovered components may reflect conformational heterogeneity at each site. For intrinsic protein fluorescence, it is often noted in the literature that single Trp proteins may be multiexponential. Genetic substitution in multi-Trp proteins, however, often leads to very strong (if not complete) lifetime-to-Trp assignment. Even if a single Trp experiences two or more microenvironments, it can be a useful reporter. The linkage of multiple lifetimes and amplitudes to changes in global conformation often reveals a more "sensitive" subpopulation or lifetime component that becomes a better indicator for important conformational states than aggregate intensity can provide. This has proven useful in studying pH transitions of proteins both in solution and embedded in membranes.