ABSTRACT
Protein stability is determined by the primary structure. As the protein unfolds, the tryptophans move away from each other, and the fluorescence intensity grows much greater. In order to determine the equilibrium constant and the free energy of folding as affected by the denaturant, a graph of fluorescence intensity versus the concentration of urea must be generated. To treat them all is far beyond the scope of a single laboratory period, for they include activity studies, calorimetry studies of multiple different types, circular dichroisms, visible and ultraviolet spectroscopy, and of course fluorescence. The intensity of fluorescence indicates how often the tryptophan gets jostled by other molecules or functional groups while in the excited form. Therefore, Fluorescence Resonance Energy Transfer experiments should best be performed at protein concentrations that can be shown not to exhibit any inner filter effects.
