Tracer diffusion of proteins in DNA solutions. 2. Green fluorescent protein in crowded DNA solutions
Tracer diffusion of proteins in DNA solutions. 2. Green fluorescent protein in crowded DNA solutions
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DOI：
10.1021/ma0005724
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发表时间：
2000-08-08
期刊：
影响因子：
5.5
通讯作者：
Bloomfield, VA
中科院分区：
文献类型：
--
作者：
Busch, NA;Kim, T;Bloomfield, VA
蛋白质在细胞内的核酸和其他大分子的拥挤、强烈相互作用的环境中扩散到它们的作用位点。为了研究在这样的环境中的一个典型的球状蛋白的动力学，我们使用荧光光漂白恢复测量探针扩散的绿色荧光蛋白（GFP）在稀释到高浓度的水溶液中的甘油，Ficoll 70，和持久长度小牛胸腺DNA。在甘油中，GFP精确地遵守将扩散系数与溶液粘度相关联的斯托克斯-爱因斯坦方程。在浓缩的Ficoll 70中，GFP的扩散速度比从粘度预测的要快，这表明在分子不均匀的溶液中存在微粘度现象。在DNA中，GFP的扩散系数明显大于从斯托克斯-爱因斯坦方程预测，在较低的离子强度与偏差增加。这种行为反映了微粘度，库仑相互作用，并在DNA溶液中的探针扩散的动力学，经历了普通的非凡的过渡，我们证明了动态光散射。
Proteins diffuse to their sites of action within cells in a crowded, strongly interacting environment of nucleic acids and other macromolecules. To investigate the dynamics of a typical globular protein in such an environment, we used fluorescence photobleaching recovery to measure the probe diffusion of green fluorescent protein (GFP) in dilute to highly concentrated aqueous solutions of glycerol, Ficoll 70, and persistence-length calf thymus DNA. In glycerol, GFP accurately obeyed the Stokes-Einstein equation that relates diffusion coefficient to solution viscosity. In concentrated Ficoll 70, GFP diffused moderately faster than predicted from viscosity, demonstrating the phenomenon of microviscosity in a molecularly heterogeneous solution. In DNA, the diffusion coefficient of GFP was markedly greater than predicted from the Stokes-Einstein equation, with deviations increasing at lower ionic strength. This behavior reflects microviscosity, Coulombic interactions, and the dynamics of probe diffusion in DNA solutions that had undergone the ordinary-extraordinary transition, which we demonstrated by dynamic Light scattering.