Department of Chemistry and Chemical Biology, and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, United States.
Biochemistry. 2012 Jan 10;51(1):100-7. doi: 10.1021/bi201362z. Epub 2011 Dec 9.
Globular proteins are usually in equilibrium with unfolded conformations, whereas kinetically stable proteins (KSPs) are conformationally trapped by their high unfolding transition state energy. Kinetic stability (KS) could allow proteins to maintain their activity under harsh conditions, increase a protein's half-life, or protect against misfolding-aggregation. Here we show the development of a simple method for quantifying a protein's KS that involves incubating a protein in SDS at high temperature as a function of time, running the unheated samples on SDS-PAGE, and quantifying the bands to determine the time-dependent loss of a protein's SDS resistance. Six diverse proteins, including two monomer, two dimers, and two tetramers, were studied by this method, and the kinetics of the loss of SDS resistance correlated linearly with their unfolding rate determined by circular dichroism. These results imply that the mechanism by which SDS denatures proteins involves conformational trapping, with a trapping rate that is determined and limited by the rate of protein unfolding. We applied the SDS trapping of proteins (S-TraP) method to superoxide dismutase (SOD) and transthyretin (TTR), which are highly KSPs with native unfolding rates that are difficult to measure by conventional spectroscopic methods. A combination of S-TraP experiments between 75 and 90 °C combined with Eyring plot analysis yielded an unfolding half-life of 70 ± 37 and 18 ± 6 days at 37 °C for SOD and TTR, respectively. The S-TraP method shown here is extremely accessible, sample-efficient, cost-effective, compatible with impure or complex samples, and will be useful for exploring the biological and pathological roles of kinetic stability.
球状蛋白通常与未折叠构象处于平衡状态，而动力学稳定的蛋白（KSP）则由于其高变性过渡态能量而被构象捕获。动力学稳定性（KS）可以使蛋白在恶劣条件下保持其活性，增加蛋白的半衰期，或防止错误折叠-聚集。在这里，我们展示了一种定量蛋白 KS 的简单方法的发展，该方法涉及将蛋白在高温下孵育在 SDS 中作为时间的函数，运行未加热的样品在 SDS-PAGE 上，并定量条带来确定蛋白对 SDS 抗性的时间依赖性丧失。通过这种方法研究了六种不同的蛋白，包括两个单体、两个二聚体和两个四聚体，SDS 抗性的丧失动力学与通过圆二色性确定的它们的变性速率呈线性相关。这些结果表明，SDS 使蛋白变性的机制涉及构象捕获，其捕获速率由蛋白变性的速率决定并受到限制。我们将 SDS 蛋白捕获（S-TraP）方法应用于超氧化物歧化酶（SOD）和转甲状腺素蛋白（TTR），它们是高度的 KSP，其天然变性速率用传统光谱方法难以测量。75 至 90°C 之间的 S-TraP 实验与 Eyring 图分析相结合，分别得出 SOD 和 TTR 在 37°C 时的变性半衰期为 70±37 和 18±6 天。这里显示的 S-TraP 方法非常易于访问、样品效率高、成本效益高、与不纯或复杂的样品兼容，并且将有助于探索动力学稳定性的生物学和病理学作用。
