Fluorescence Quenching Methods to Study Protein–Nucleic Acid Interactions

Abstract

The study of protein–nucleic acid interaction is at the heart of many biological phenomena. Dissociation constants of protein–nucleic acid complexes range from 10-12M for lac repressor–operator interaction to 10-4M for some tRNA–synthetase interactions and stoichiometry ranges from many proteins binding to a single ligand, as in the case of DNA-binding proteins to several ligands binding to a single protein, as in the case tRNA binding to proteins. No single technique or strategy is suitable for studying such diversity. Many methods have been devised, such as quantitative footprinting, optical spectroscopy, nuclear magnetic resonance (NMR), electrophoresis, and ultracentrifugation, to name a few. Each technique has its own advantages and disadvantages and the range of dissociation constant in which it works best. Fluorescence spectroscopy has been widely used to study ligand–protein interaction because of its simplicity and sensitivity. Fluorescence methods are particularly popular because they are well suited for protein–ligand interactions with dissociation constants in the range of 10-4 to 10-8M, where some protein–nucleic acid dissociation constants fall. It is difficult to measure the intrinsic fluorescence of proteins reliably at a concentration lower than 10-8M. Hence, it is difficult to measure dissociation constants in the subnanomolar range, where many operator–repressor interactions fall. Many fluorescence parameters, such as anisotropy, intensity, and energy transfer efficiency, are sensitive to formation of protein–ligand complexes and can be utilized to derive binding isotherms. The change in fluorescence intensity on formation of a protein–ligand complex is one of the simplest ways to measure binding of a ligand to a protein. It offers a way to estimate the interaction free energy at equilibrium without any special assumptions. Such equilibrium measurements are vital for obtaining thermodynamic parameters. In addition to obtaining equilibrium constants, fluorescence quenching methods can provide clues to conformation of the protein–nucleic acid complexes. External collisional quenchers offer another way to detect conformational differences of protein–nucleic acid complexes. We divide the chapter into two parts: the first deals with issues related to the measurement of binding affinity and the second deals with the elucidation of conformation of the nucleic acid-bound proteins.