In a recent article, we described the application of phasor analysis to fluorescence intensity decay data on in vitro samples. As detailed in that article, this method provides researchers with a simple graphical method for viewing lifetime data that can be used to quantify individual components of a mixture as well as to identify excited state reactions. In the current article, we extend the use of in vitro phasor analysis to intrinsic protein fluorescence. We show how alterations in the excited state properties of tryptophan residues are easily visualized using the phasor method. Specifically, we demonstrate that protein-ligand and protein-protein interactions can result in unique shifts in the location of phasor points, indicative of protein conformational changes. Application of the method to a rapid kinetic experiment is also shown. Finally, we show that the unfolding of lysozyme with either urea or guanidine hydrochloride results in different phasor trajectories, indicative of unique denaturation pathways.
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