Fluorescence anisotropy and resonance energy transfer: powerful tools for measuring real time protein dynamics in a physiological environment

Abstract

Fluorescence spectroscopy/microscopy is a versatile method for examining protein dynamics in vitro and in vivo that can be combined with other techniques to simultaneously examine complementary pharmacological parameters. The following review will highlight the advantages and challenges of using fluorescence spectroscopic methods for examining protein dynamics with a special emphasis on fluorescence resonance energy transfer and fluorescence anisotropy. Both of these methods are amenable to measurements on an ensemble of molecules as well as at the single molecule level, in live cells and in high throughput screening assays, providing a powerful set of tools to aid in the design and testing of new drugs under a variety of experimental conditions.

Figure 1

Summary of hypothetical FRET and FA measurements on a protein that changes conformation upon binding to a ligand. A) FRET can be performed by examining the steady-state fluorescence quenching of the donor in the presence of the acceptor and/or measuring the enhancement in the acceptor fluorescence in the presence of the donor. Time-resolved studies monitor donor lifetime in the presence and absence of the acceptor to obtain distance distributions in each state. B) Anisotropy measurements are examined by monitoring the vertical and horizontal components of the emitted fluorescence following excitation with vertically polarized light. The rotational correlation time (θ) and the angle of rotation within a cone, defined by r_∞, can be determined providing information about the dynamics of the structural elements or the entire protein. Anisotropy is highly sensitive to formation of higher order oligomers or protein complexes. The inset demonstrates the steady-state anisotropy of the labeled protein in the presence of different concentrations of ligand which can be used to measure the binding affinity of the protein-ligand complex.