Andrews Jennifer M, Roberts Christopher J
Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA.
Biochemistry. 2007 Jun 26;46(25):7558-71. doi: 10.1021/bi700296f. Epub 2007 May 27.
The kinetics and structural transitions of non-native aggregation of alpha-chymotrypsinogen (aCgn) were investigated over a wide range of temperature and initial protein concentration at pH 3.5, where high molecular weight aggregates remained soluble throughout the reaction. A comparison of thermodynamic, kinetic, and spectroscopic data shows that aggregation under non-native-favoring conditions proceeds through a molten globule unfolded monomer state, with a nucleation and growth mechanism. Formation of irreversible aggregates and conversion to beta-sheet secondary structures occur simultaneously without detectable intermediates, suggesting that beta-sheet formation may be a commitment step during the nucleation and growth stages. Analysis of the kinetics using a Lumry-Eyring with nucleated polymerization (LENP) model provides the predominant nucleus size and the product of the intrinsic nucleation and intrinsic growth time scales at each state point. We find that the nucleus size depends on both temperature and protein concentration, and in some cases there is competition between two distinct nucleus sizes. The observed rate coefficient (kobs) for aggregation displays a maximum as a function of temperature because of the competition between folding-unfolding thermodynamics and the intrinsic growth and nucleation rates; the latter contribution has a large, negative activation enthalpy that dominates kobs at elevated temperatures. Temperature-jump experiments reveal that aggregates depolymerize at high temperatures, indicating that they are lower in enthalpy than the free monomer. Overall, the results suggest more generally that non-native aggregation may proceed through more than one nucleus size and that intrinsic kinetics of nucleation and growth may have significant entropic barriers.
在pH 3.5的条件下，研究了α-胰凝乳蛋白酶原（aCgn）在广泛的温度和初始蛋白质浓度范围内非天然聚集的动力学和结构转变，在此条件下，高分子量聚集体在整个反应过程中保持可溶。对热力学、动力学和光谱数据的比较表明，在有利于非天然构象的条件下，聚集过程通过熔融球状展开单体状态进行，具有成核和生长机制。不可逆聚集体的形成和向β-折叠二级结构的转变同时发生，没有可检测到的中间体，这表明β-折叠的形成可能是成核和生长阶段的一个决定性步骤。使用带有成核聚合的Lumry-Eyring（LENP）模型对动力学进行分析，可得出每个状态点的主要核尺寸以及固有成核和固有生长时间尺度的乘积。我们发现核尺寸取决于温度和蛋白质浓度，在某些情况下，两种不同的核尺寸之间存在竞争。由于折叠-展开热力学与固有生长和成核速率之间的竞争，观察到的聚集速率系数（kobs）随温度呈现最大值；后者的贡献具有很大的负活化焓，在高温下主导kobs。温度跳跃实验表明，聚集体在高温下解聚，这表明它们的焓比游离单体低。总体而言，结果更普遍地表明，非天然聚集可能通过不止一种核尺寸进行，并且成核和生长的固有动力学可能具有显著的熵垒。
