Effect of crowding on protein - Protein association rates: Fundamental differences between low and high mass crowding agents
Effect of crowding on protein - Protein association rates: Fundamental differences between low and high mass crowding agents
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DOI：
10.1016/j.jmb.2003.12.008
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发表时间：
2004-02-20
影响因子：
5.6
通讯作者：
Schreiber, G
中科院分区：
文献类型：
--
作者：
Kozer, N;Schreiber, G
生理介质构成了一个拥挤的环境，作为体内蛋白质-蛋白质相互作用的作用场。在拥挤的溶液中测量蛋白质-蛋白质相互作用可以模拟这种环境。在这项工作中，我们遵循的过程中的蛋白质-蛋白质协会和它的速率常数（k（on））的β-内酰胺酶（TEM）-β-内酰胺酶抑制剂蛋白（BLIP）复合物在拥挤的溶液中使用低和高分子量拥挤剂。在所有拥挤的溶液（0-40%（w/w）的乙二醇（EG）、聚（乙二醇）（PEG）200、1000、3350、8000 Da Ficoll-70和Haemaccel）中，发现与缓冲液相比，测量的绝对k（开）而不是k（关）值较慢。然而，低质量和高质量拥挤剂之间存在根本区别。在低质量拥挤剂和海脉素的存在下，k（on）与溶液粘度成反比。在高质量聚合物溶液中，即使在粘度比水高12倍的情况下，k（on）也仅轻微变化。低分子量聚合物和高分子量聚合物之间的边界是尖锐的，并且由聚合物长度（L）与其持久长度（L-p）之间的比率决定。长到足以形成柔性线圈的聚合物（L/L-p > 2）表现为高分子量聚合物，而不能形成柔性线圈的聚合物（L/L-p < 2）表现为低分子量聚合物。我们的结论是，虽然聚合物溶液是拥挤的，但这种性质是不均匀的;即，在溶液中存在含有大量水的区域，并且在这些区域中，蛋白质可以扩散和缔合，几乎就像它们在稀释的环境中一样。这种多孔介质可以被视为模仿细胞环境的某些方面，其中许多大分子沿沿着膜和细胞骨架组织。为了确定拥挤环境中蛋白质之间的静电吸引的贡献，我们跟踪了wt-TEM和三种BLIP类似物的k（on），由于静电转向，k（on）值增加了100倍。更快的关联BLIP变体在所有拥挤的解决方案中保持其相对优势，包括Haemaccel。这一结果表明，快速缔合蛋白质复合物在复杂环境中也保持了其优势。(C)2003 Elsevier Ltd.保留所有权利。
Physiological media constitutes a crowded environment that serves as the field of action for protein-protein interaction in vivo. Measuring protein-protein interaction in crowded solutions can mimic this environment. In this work we follow the process of protein-protein association and its rate constants (k(on)) of the beta-lactamase (TEM)-beta-lactamase inhibitor protein (BLIP) complex in crowded solution using both low and high molecular mass crowding agents. In all crowded solutions (0-40% (w/w) of ethylene glycol (EG), poly(ethylene glycol) (PEG) 200, 1000, 3350, 8000 Da Ficoll-70 and Haemaccel the measured absolute k(on), but not k(off) values, were found to be slower as compared to buffer. However, there is a fundamental difference between low and high mass crowding agents. In the presence of low mass crowding agents and Haemaccel k(on) depends inversely on the solution viscosity. In high mass polymer solutions k(on) changes only slightly, even at viscosities 12-fold higher than water. The border between low and high molecular mass polymers is sharp and is dictated by the ratio between the polymer length (L) and its persistence length (L-p). Polymers that are long enough to form a flexible coil (L/L-p > 2) behave as high molecular mass polymers and those who are unable to do so (L/L-p < 2) behave as low molecular mass polymers. We concluded that although polymers solution are crowded, this property is not uniform; i.e. there are areas in the solution that contain bulk water, and in these areas proteins can diffuse and associate almost as if they were in diluted environment. This porous medium may be taken as mimicking some aspects of the cellular environment, where many of the macromolecules are organized along membranes and the cytoskeleton. To determine the contribution of electrostatic attraction between proteins in crowded milieu, we followed k(on) of wt-TEM and three BLIP analogs with up to 100-fold increased values of k(on) due to electrostatic steering. Faster associating BLIP variants keep their relative advantage in all crowded solutions, including Haemaccel. This result suggests that faster associating protein complexes keep their advantage also in complex environment. (C) 2003 Elsevier Ltd. All rights reserved.