Special ArticleFluorescence Resonance Energy Transfer Spectroscopy Is a Reliable "Ruler" for Measuring Structural Changes in Proteins: Dispelling the Problem of the Unknown Orientation Factor
Abstract
Fluorescence resonance energy transfer (FRET) spectroscopy has been widely used to "measure" dimensions either within or between molecules over distances of 10-100Å, a range that is well suited to probing protein structure. The resolution of FRET spectroscopy is substantially lower than X-ray diffraction (XRD) but the technique makes up for this deficit by being particularly good at measuring structural changes in proteins. However, absolute distances measured by FRET spectroscopy remain problematical because of what appears to be an unjustified assumption, namely that calculations of FRET distances assume that the probes are able to undergo free, isotropic motion. This uncertainty may be ascribed to an unknown value for the orientation factor, but other factors may also be important. Common sense suggests that a large (300-500 Da) hydrophobic FRET probe covalently bound to an amino acid side chain of a protein can not undergo true rotational freedom. If this is so, the calculated distances would at best be flawed and at worst be meaningless. In this paper we argue that the orientation parameter is no longer an important issue in the determination of distances determined by FRET using peptides and proteins. Furthermore, we suggest that FRET may be a good form of spectroscopy for testing models of F-actin.
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