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Fluorescense Anisotropy

Fluorescence anisotropy is a method for measuring the binding interaction between two molecules, and can be used to measure the binding constant (or the inverse, the disassociation constant) for the interaction. The basic idea is that a fluorophore excited by polarized light (light whose "waves" only go one direction) will also emit polarized light. However, if a molecule is moving, it will tend to "scramble" the polarization of the light by radiating at a different direction from the incident light. The "scrambling" effect is greatest with fluorophores freely tumbling in solution and decreases with decreased rates of tumbling. Protein interactions can be detected when one of the interacting partners is fused to a fluorophore: upon binding of the partner molecule a larger, more stable complex is formed which will tumble more slowly (thus, increasing the polarization of the emitted light and reducing the "scrambling" effect). This technique works best if a small molecule is fused to a fluorophore and binds to a larger partner (this maximizes the difference in signal between bound and unbound states). If the fluorophore is attached to the larger protein in a binding pair, the difference in polarization between bound and unbound states will be smaller (because the unbound protein will already be fairly stable and tumble slowly to begin with) and the measurement will be less precise.

By titrating the amount of one of the proteins, a binding curve can be generated (the amount of polarization observed is proportional to the amount of protein complex formed, which is proportional to the concentration of the binding partners in solution). Mathematical models can be applied to this binding curve to determine the binding constant of the protein interaction. In another application of this technique, it is also possible to measure the folding of a protein, since an unfolded peptide chain will tumble differently than a folded one, giving a difference in polarization over time. This provides a measure of the dynamics of how the protein achieves its final, stable 3D shape.

Editor(s): John Bracht

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