Protein aggregation is one of the most critical processes affecting protein solubility in various contexts—from protein therapeutics formulation to protein diseases. In general, time‐dependent changes in protein solubility are complex kinetically driven processes that often involve a triggering event that consists of a protein unfolding/misfolding followed by the assembling of aggregation‐competent protein species. In this study, we have examined the relation between stability and time‐dependent solubility of the recombinant human antibody light chain, hLC, which was found to form renal tubular casts in the multiple myeloma patient. To analyze the aggregation quantitatively, the hLC stability and protein solubility assays were performed in vitro at elevated temperatures. A differential acceleration of the processes at high temperatures enabled us to dissect observed kinetics of irreversible hLC unfolding and aggregation. We find that for hLC these processes have different molecularity and activation energy barriers. While the irreversible unfolding of hLC is a unimolecular step with a substantial activation energy barrier of 260 kJ/mol, the aggregation is rate‐limited by the bimolecular reaction, which is characterized by a lower activation energy barrier of 40 kJ/mol. By the combination of experimental assays at different temperatures, different protein concentrations and kinetic modeling using ordinary differential equations, we were able to extrapolate time‐dependent protein solubility to temperatures where both unfolding and aggregation processes are strongly kinetically coupled. Our study enables mechanism‐based evaluation and interpretation of different physico‐chemical factors contributing to the hLC unfolding and aggregation and their effect on the formation of extracellular protein deposits.

中文翻译：

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蛋白质解折叠和聚集之间的动力学耦合控制人骨髓瘤抗体轻链的时间依赖性溶解度

蛋白质聚集是在各种情况下影响蛋白质溶解度的最关键过程之一——从蛋白质治疗剂配方到蛋白质疾病。一般来说，蛋白质溶解度的时间依赖性变化是复杂的动力学驱动过程，通常涉及触发事件，该事件包括蛋白质解折叠/错误折叠，然后是具有聚集能力的蛋白质种类的组装。在这项研究中，我们检查了重组人抗体轻链 hLC 的稳定性和时间依赖性溶解度之间的关系，发现该轻链在多发性骨髓瘤患者中形成肾小管管型。为了定量分析聚集，在体外在升高的温度下进行 hLC 稳定性和蛋白质溶解度测定。高温下过程的不同加速使我们能够剖析观察到的不可逆 hLC 展开和聚集的动力学。我们发现对于 hLC，这些过程具有不同的分子和活化能垒。虽然 hLC 的不可逆解折叠是一个单分子步骤，活化能垒为 260 kJ/mol，但聚集受双分子反应限速，其特点是活化能垒较低，为 40 kJ/mol。通过结合不同温度、不同蛋白质浓度和使用常微分方程的动力学建模的实验分析，我们能够将时间依赖性蛋白质溶解度外推到解折叠和聚集过程都强烈动力学耦合的温度。