Fluorescence anisotropy peptide-protein interactions

Yang, P., Whelan, R. J., Jameson, E. E., Kurzer, J. H., Argetsinger, L. S., Carter-Su, C., Kabir, A., Malik, A., Kennedy, R. T. (2005). Capillary electrophoresis and fluorescence anisotropy for quantitative analysis of peptide-protein interactions using JAK2 and SH2-BP as a model system. Anal Chem 11, 2482-2489. 

APCE has also been used to investigate the fast kinetics of peptide-protein interactions. A fluorescently labeled phosphorylated peptide was mixed with a Src homology 2-domain protein and separations of complex and free probe could be achieved in less than 4 s. These rapid separations were necessary, because no complex peak was observed with 8 s separations, presumably because it had dissociated. Estimated dissociation constants for the complex were obtained from peak areas and results were similar to constants from fluorescence anisotropy data. Aptamers have also been used as affinity ligands for fast separations. Separations as short as 30 s were accomplished with a fluorescently labeled aptamer against IgE. " APCE has also been used to detect nM concentrations of digoxin in less than 1 min. ... 

Yang, P. et al. Capillary electrophoresis and fluorescence anisotropy for quantitative analysis of peptide-protein interactions using JAK2 and SH2-beta as a model system. Anal. Chem. 77, 2482, 2005. 

The possibility to carry out conformational studies of peptides at low concentrations and in the presence of complex biological systems represents a major advantage of fluorescence spectroscopy over other techniques. Fluorescence quantum yield or lifetime determinations, anisotropy measurements and singlet-singlet resonance energy transfer experiments can be used to study the interaction of peptides with lipid micelles, membranes, proteins, or receptors. These fluorescence techniques can be used to determine binding parameters and to elucidate conformational aspects of the interaction of the peptide with a particular macro-molecular system. The limited scope of this chapter does not permit a comprehensive review of the numerous studies of this kind that have been carried and only a few general aspects are briefly discussed here. Fluorescence studies of peptide interactions with macromolecular systems published prior to 1984 have been reviewed. 

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