Polyvinylpyrrolidone 40 assists the refolding of bovine carbonic anhydrase B by accelerating the refolding of the first molten globule intermediate
Polyvinylpyrrolidone 40 assists the refolding of bovine carbonic anhydrase B by accelerating the refolding of the first molten globule intermediate
复制标题
聚乙烯吡咯烷酮 40 通过加速第一个熔球中间体的重折叠来协助牛碳酸酐酶 B 的重折叠
DOI：
10.1074/jbc.m507874200
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发表时间：
2006-04-07
影响因子：
4.8
通讯作者：
Zhou, HM
中科院分区：
文献类型：
--
作者：
Jiang, Y;Yan, YB;Zhou, HM
保护蛋白质免于聚集是蛋白质科学和蛋白质工程中最重要的问题之一。本文通过动力学和平衡复性实验研究了聚乙烯吡咯烷酮40（PVP 40）促进盐酸胍变性碳酸酐酶B复性的机理。再活化和复性动力学研究表明，PVP的加入提高了第一个复性中间体（I-1）复性为第二个复性中间体（I-2）的速率常数。荧光猝灭研究进一步表明，PVP可以结合到易于聚集的物种I-1，导致暴露的疏水表面的保护，最小化的蛋白质表面，更重要的是，增加的I-1的复性速率。这些性质是完全不同的聚（乙二醇）（PEG），已被证明有一个强大的和化学计量的结合到I-1，并不干扰的重折叠途径。与PEG不同，PVP与I-1的结合并不直接阻断聚集途径，而是降低了I-1重折叠为I-2的能垒，从而减少了I-1的积累。这些结果表明PVP的作用机制与分子伴侣和化学促进剂的作用机制完全不同。PVP更像是一种折叠催化剂，而不是化学伴侣。PVP增强蛋白质聚集的独特机制有望促进开发新化合物以及保护蛋白质免于聚集的新策略的尝试。
Protecting proteins from aggregation is one of the most important issues in both protein science and protein engineering. In this research, the mechanism of enhancing the refolding of guanidine hydrochloride-denatured carbonic anhydrase B by polyvinylpyrrolidone 40 (PVP40) was studied by both kinetic and equilibrium refolding experiments. The reactivation and refolding kinetics indicated that the rate constant of refolding the first refolding intermediate (I-1) to the second one (I-2) is promoted by the addition of PVP. Fluorescence quenching studies further indicated that PVP could bind to the aggregation-prone species I-1, resulting in the protection of the exposed hydrophobic surface, a minimization of the protein surface, and more importantly, an increase of the refolding rate of I-1. These properties were quite different from those of poly(ethylene glycol) (PEG), which has been shown to have a strong and stoichiometric binding to I-1 and does not interfere with the refolding pathway. Unlike PEG, the binding of PVP to I-1 does not block the aggregation pathway directly but decreases the energy barrier for I-1 to refold to I-2 and thus reduces the accumulation of I-1. These results suggested that PVP works by a quite different mechanism from those well established ones in chaperones and chemical promoters. PVP is more like a folding catalyst rather than a chemical chaperone. The distinct mechanism of enhancing protein aggregation by PVP is expected to facilitate the attempt to develop new chemical compounds as well as new strategies to protect proteins from aggregation.