Optical studies of molecular motions: using fluorescence anisotropy decays to determine the shapes of dye molecules, proteins, and nucleosomes

Abstract

Molecules undergo Brownian rotational diffusion, continually tumbling through solution. If a molecule contains a fluorescent chromophore with an appropriate excited-state lifetime, and a solution of such molecules is excited with a brief flash of polarized laser light, then it is possible to measure the rates at which the molecule tumbles. One can measure the anisotropy of the decay of the fluorescence, a measure of the depolarization of the fluorescence in time. The rates of rotational diffusion are sensitive to the shape of the molecule. This paper examines the molecular shape information available from the anisotropy decay. Three systems, which are of interest to our laboratory, are described here. Our studies on the dye rose bengal (molecular weight ~1000) suggest that an approximation used in rotational diffusion theory-that the solvent molecules are very small compared to the solute-is valid even for this relatively small molecule. Next, we examine the effects of calcium concentration on the rotational diffusion ofthe protein calmodulin (molecular weight ~17,000) derivatized by photoinduced crosslinking to form a dityrosine fluorophore. In the presence of sufficiently high calcium ion concentration, this crosslinked calmodulin shows a single exponential anisotropy decay consistent with the rotational diffusion of the extended dumbbell structure found for calmodulin by x-ray crystallography. At low calcium concentrations, the crosslinked calmodulin rotates considerably faster, suggesting a much more compact shape; also, this anisotropy decay shows short correlation times that are interpreted as arising from a segmental flexibility not evident for crosslinked calmodulin at high calcium concentration. Finally, we examine a transition that is observed at very low salt concentrations for nucleosome core particles, relatively large complexes (molecular weight ~204,000) derived from the chromatin of higher organisms and comprised of eight histone molecules and 145 base pairs of DNA.