肌钙蛋白C突变体F22W在单光子、双光子和三光子激发下的荧光光谱特性。
Fluorescence spectral properties of troponin C mutant F22W with one-, two-, and three-photon excitation.
作者信息
Gryczynski I, Malak H, Lakowicz J R, Cheung H C, Robinson J, Umeda P K
机构信息
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore 21201, USA.
出版信息
Biophys J. 1996 Dec;71(6):3448-53. doi: 10.1016/S0006-3495(96)79540-1.
We report the first measurements of protein fluorescence with three-photon excitation, using a mutant of troponin C (TnC) that contains a single tryptophan residue F22W. From the emission intensity dependence on laser power we determine that TnC F22W displays one-, two-, and three-photon excitation at 285, 570, and 855 nm, respectively. The emission spectra and intensity decays are identical for one-, two-, or three-photon excitation. The steady-state and time 0 anisotropies are distinct for each mode of excitation, but the correlation times were the same, suggesting that three-photon excitation of proteins can be accomplished without significant effects of the locally intense illumination. The excitation anisotropy spectrum from 830 to 900 nm displays only negative values, suggesting dominant excitation via the 1Lb state of tryptophan from 830 to 900 nm.
我们报道了首次使用含有单个色氨酸残基F22W的肌钙蛋白C（TnC）突变体进行三光子激发的蛋白质荧光测量。根据发射强度对激光功率的依赖性，我们确定TnC F22W分别在285、570和855 nm处表现出单光子、双光子和三光子激发。单光子、双光子或三光子激发的发射光谱和强度衰减是相同的。每种激发模式的稳态和时间0各向异性是不同的，但相关时间是相同的，这表明蛋白质的三光子激发可以在不受到局部强光显著影响的情况下实现。830至900 nm的激发各向异性光谱仅显示负值，表明在830至900 nm范围内主要通过色氨酸的1Lb态进行激发。
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