Enhanced resolution of fluorescence anisotropy decays by simultaneous analysis of progressively quenched samples. Applications to anisotropic rotations and to protein dynamics.
Enhanced resolution of fluorescence anisotropy decays by simultaneous analysis of progressively quenched samples. Applications to anisotropic rotations and to protein dynamics.
复制标题
通过同时分析逐渐淬灭的样品，增强荧光各向异性衰减的分辨率。
DOI：
10.1016/s0006-3495(87)83402-1
复制
发表时间：
1987
影响因子：
3.4
通讯作者：
Johnson,ML
中科院分区：
文献类型：
--
作者：
Lakowicz,JR;Cherek,H;Gryczynski,I;Joshi,N;Johnson,ML
通过测量和分析逐渐淬火样品的数据，获得了快速和复杂的各向异性衰减的增强分辨率。丙烯酰胺的碰撞猝灭被用来改变吲哚或蜂毒肽的色氨酸荧光的平均衰减时间。从极化发射在4至2,000 MHz频率的频率响应获得各向异性衰减。猝灭增加了总发射的分数，这发生在亚纳秒时间尺度上，从而提供了更多的信息皮秒旋转运动或蛋白质的局部运动。对于单指数亚纳秒各向异性衰减，增强的分辨率是通过测量最高淬火样品。对于复杂的各向异性衰减，如由于局部运动和整体蛋白质旋转扩散，上级分辨率获得淬火和未淬火样品的数据的同时分析。我们表明，淬火样品的测量大大降低了吲哚在水中在20摄氏度的50-ps相关时间的不确定性，并允许吲哚的各向异性旋转的相关时间为140和720 ps的分辨率。该方法适用于蜂毒素的单体和四聚体形式。随着淬灭的增加，各向异性数据显示整体蛋白质旋转的贡献减少，皮秒色氨酸运动的贡献增加。在单体和四聚体形式的蜂毒肽的色氨酸残基显示大量的本地运动与相关时间分别接近0.16和0.06纳秒。在四聚体中，局部运动的幅度小两倍。这些高分辨率的各向异性衰减应该是有价值的蜂毒素的分子动力学模拟比较。
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.