Laboratory of Biochemistry, Microspectroscopy Centre, Wageningen University, PO Box 8128, 6700 ET Wageningen, The Netherlands.
Methods Appl Fluoresc. 2016 Aug 18;4(3):035002. doi: 10.1088/2050-6120/4/3/035002.
The molecular dimensions of proteins such as green fluorescent protein (GFP) are large as compared to the ones of solvents like water or glycerol. The microscopic viscosity, which determines the resistance to diffusion of, e.g. GFP, is then the same as that determined from the resistance of the solvent to flow, which is known as macroscopic viscosity. GFP in water/glycerol mixtures senses this macroscopic viscosity, because the translational and rotational diffusion coefficients are proportional to the reciprocal value of the viscosity as predicted by the Stokes-Einstein equations. To test this hypothesis, we have performed time-resolved fluorescence anisotropy (reporting on rotational diffusion) and fluorescence correlation spectroscopy (reporting on translational diffusion) experiments of GFP in water/glycerol mixtures. When the solvent also contains macromolecules of similar or larger dimensions as GFP, the microscopic and macroscopic viscosities can be markedly different and the Stokes-Einstein relations must be adapted. It was established from previous dynamic fluorescence spectroscopy observations of diffusing proteins with dextran polysaccharides as co-solvents (Lavalette et al 2006 Eur. Biophys. J. 35 517-22), that rotation and translation sense a different microscopic viscosity, in which the one arising from rotation is always less than that from translation. A microscopic viscosity parameter is defined that depends on scaling factors between GFP and its immediate environment. The direct consequence is discussed for two reported diffusion coefficients of GFP in living cells.
与水或甘油等溶剂相比，绿色荧光蛋白（GFP）等蛋白质的分子尺寸较大。决定 GFP 等扩散阻力的微观粘度与溶剂流动阻力（即宏观粘度）相同。GFP 在水/甘油混合物中感知这种宏观粘度，因为根据 Stokes-Einstein 方程的预测，平移和旋转扩散系数与粘度的倒数成正比。为了验证这一假设，我们对 GFP 在水/甘油混合物中的时间分辨荧光各向异性（报告旋转扩散）和荧光相关光谱（报告平移扩散）实验进行了研究。当溶剂中也含有与 GFP 具有相似或更大尺寸的大分子时，微观和宏观粘度可能明显不同，Stokes-Einstein 关系必须进行调整。以前用葡聚糖多糖作为共溶剂的扩散蛋白的动态荧光光谱观察（Lavalette 等人，2006 年，Eur. Biophys. J. 35 517-22）已经确定，旋转和平移感知到不同的微观粘度，其中旋转产生的粘度总是小于平移产生的粘度。定义了一个微观粘度参数，该参数取决于 GFP 与其直接环境之间的比例因子。对于两个报道的 GFP 在活细胞中的扩散系数，讨论了直接后果。
