Analysis of DNA-Protein Interactions by Intrinsic Fluorescence

Changes in the fluorescence emission spectrum of a protein upon binding to DNA can often be used to determine the stoichiometry of binding and equilibrium binding constants; in some cases the data can also give an indication of the location of particular residues within the protein. The experiments are generally quick and easy to perform, requiring only small quantities of material (1). Spectroscopic techniques allow one to measure binding at equilibrium (unlike, for example, gel retardation assays and other separation techniques that are strictly nonequilibrium methods). Fluorescence is one of the most sensitive of spectroscopic techniques, allowing the low concentrations (typically in the nanomolar to micromolar range) required for estimation of binding constants for many protein-DNA interactions. Considerable care, however, needs to be exercised in the experiment itself and in the interpretation of results. The fundamental principles of fluorescence are discussed briefly in the remainder of the Introduction.