Enhanced resolution of fluorescence anisotropy decays by simultaneous analysis of progressively quenched samples. Applications to anisotropic rotations and to protein dynamics.
Enhanced resolution of rapid and complex anisotropy decays was obtained by measurement and analysis of data from progressively quenched samples. Collisional quenching by acrylamide was used to vary the mean decay time of indole or of the tryptophan fluorescence from melittin. Anisotropy decays were obtained from the frequency-response of the polarized emission at frequencies from 4 to 2,000 MHz. Quenching increases the fraction of the total emission, which occurs on the subnanosecond timescale, and thereby provides increased information on picosecond rotational motions or local motions in proteins. For monoexponential subnanosecond anisotropy decays, enhanced resolution is obtained by measurement of the most highly quenched samples. For complex anisotropy decays, such as those due to both local motions and overall protein rotational diffusion, superior resolution is obtained by simultaneous analysis of data from quenched and unquenched samples. We demonstrate that measurement of quenched samples greatly reduces the uncertainty of the 50-ps correlation time of indole in water at 20 degrees C, and allows resolution of the anisotropic rotation of indole with correlation times of 140 and 720 ps. The method was applied to melittin in the monomeric and tetrameric forms. With increased quenching, the anisotropy data showed decreasing contributions from overall protein rotation and increased contribution from picosecond tryptophan motions. The tryptophan residues in both the monomeric and the tetrameric forms of melittin displayed substantial local motions with correlation times near 0.16 and 0.06 ns, respectively. The amplitude of the local motion is twofold less in the tetramer. These highly resolved anisotropy decays should be valuable for comparison with molecular dynamics simulations of melittin.
D020106:Acrylamide; D000178:Acrylamides; D001514:Bee Venoms; D005454:Fluorescence Polarization; D007211:Indoles; D008555:Melitten; D019946:Propylene Glycol; D011409:Propylene Glycols; D011487:Protein Conformation; D014364:Tryptophan
D000178:Acrylamides; D001514:Bee Venoms; D007211:Indoles; D011409:Propylene Glycols; D008555:Melitten; D020106:Acrylamide; D019946:Propylene Glycol; C030374:indole; D014364:Tryptophan