Monitoring protein aggregation during thermal unfolding in circular dichroism experiments
Monitoring protein aggregation during thermal unfolding in circular dichroism experiments
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DOI：
10.1110/ps.051917406
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发表时间：
2006-03-01
期刊：
影响因子：
8
通讯作者：
Gursky, O
中科院分区：
文献类型：
--
作者：
Benjwal, S;Verma, S;Gursky, O
通过光谱学或量热法监测的热解折叠被广泛用于确定蛋白质稳定性。这种去折叠的平衡热力学分析常常因其不可逆性而受到阻碍，这种不可逆性通常是由热变性蛋白质的聚集造成的。此外，热诱导的蛋白质错误折叠和聚集常常导致淀粉样蛋白样结构的形成。我们提出了一种通过在圆二色性 (CD) 光谱实验中记录浊度或 90 度光散射数据来实时监测热折叠/解折叠转变过程中蛋白质聚集的便捷方法。由于浊度和 90 度光散射的测量可以与远紫外或近紫外 CD 数据收集同时进行，因此不需要额外的时间或样品，并且可以与 CD 监测的蛋白质构象变化直接相关。这些结果可以为不可逆构象变化的起源提供有用的见解，并测试各种大分子系统中蛋白质解折叠或错误折叠和聚集之间的联系，包括本研究中描述的球状蛋白质和蛋白质-脂质复合物，以及各种淀粉样蛋白形成蛋白质和肽。
Thermal unfolding monitored by spectroscopy or calorimetry is widely used to determine protein stability. Equilibrium thermodynamic analysis of such unfolding is often hampered by its irreversibility, which usually results from aggregation of thermally denatured protein. In addition, heat-induced protein misfolding and aggregation often lead to formation of amyloid-like structures. We propose a convenient method to monitor in real time protein aggregation during thermal folding/unfolding transition by recording turbidity or 90 degrees light scattering data in circular dichroism (CD) spectroscopic experiments. Since the measurements of turbidity and 90 degrees light scattering can be done simultaneously with far- or near-UV CD data collection, they require no additional time or sample and can be directly correlated with the protein conformational changes monitored by CD. The results can provide useful insights into the origins of irreversible conformational changes and test the linkage between protein unfolding or misfolding and aggregation in various macromolecular systems, including globular proteins and protein-lipid complexes described in this study, as well as a wide range of amyloid-forming proteins and peptides.