Thibeault Jane, Church Jennifer, Ortiz-Perez Brian, Addo Samuel, Hill Shakeema, Khalil Areeg, Young Malaney, Xia Ke, Colón Wilfredo
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Biochemistry and Biophysics Graduate Program, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; The Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Biochem Biophys Res Commun. 2017 Sep 30;491(4):994-999. doi: 10.1016/j.bbrc.2017.07.166. Epub 2017 Aug 1.
In common beans and lima bean, the storage protein phaseolin is difficult to degrade and SDS-resistant, a sign of kinetic stability. Kinetically stable proteins (KSPs) are characterized by having a high-energy barrier between the native and denatured states that results in very slow unfolding. Such proteins are resistant to proteolytic degradation and detergents, such as SDS. Here the method SDS-Trapping of Proteins (S-TraP) is applied directly on bean extracts to quantify the kinetic stability of phaseolin in lima bean and several common beans, including black bean, navy bean, and small red bean. The bean extracts were incubated in SDS at various temperatures (60-75 °C) for different time periods, followed by SDS-PAGE analysis at room temperature, and subsequent band quantification to determine the kinetics of phaseolin unfolding. Eyring plot analysis showed that the phaseolin from each bean has high kinetic stability, with an SDS-trapping (i.e. unfolding) half-life ranging from about 20-100 years at 24 °C and 2-7 years at 37 °C. The remarkably high kinetic stability of these phaseolin proteins is consistent with the low digestibility of common beans and lima bean, as well as their relatively high germination temperatures. From a practical perspective, this work exemplifies that S-TraP is a useful and cost-effective method for quantifying the kinetic stability of proteins in biological extracts or lysates. Depending on the protein to be studied and its abundance, S-TraP may be performed directly on the extract without need for protein purification.
在菜豆和利马豆中，储存蛋白菜豆蛋白难以降解且具有抗十二烷基硫酸钠（SDS）的特性，这是动力学稳定性的一种表现。动力学稳定蛋白（KSPs）的特点是在天然态和变性态之间存在高能垒，导致其解折叠非常缓慢。这类蛋白对蛋白水解降解和去污剂（如SDS）具有抗性。在此，蛋白SDS捕获法（S-TraP）直接应用于豆类提取物，以量化利马豆和几种常见菜豆（包括黑豆、海军豆和小红豆）中菜豆蛋白的动力学稳定性。将豆类提取物在不同温度（60 - 75°C）下于SDS中孵育不同时间段，随后在室温下进行SDS聚丙烯酰胺凝胶电泳（SDS-PAGE）分析，并进行后续条带定量以确定菜豆蛋白解折叠的动力学。艾林方程分析表明，每种豆类中的菜豆蛋白都具有较高的动力学稳定性，在24°C时SDS捕获（即解折叠）半衰期约为20 - 100年，在37°C时为2 - 7年。这些菜豆蛋白极高的动力学稳定性与菜豆和利马豆的低消化率以及它们相对较高的发芽温度相一致。从实际角度来看，这项工作例证了S-TraP是一种用于量化生物提取物或裂解物中蛋白质动力学稳定性的有用且经济高效的方法。根据待研究的蛋白质及其丰度，S-TraP可直接在提取物上进行，无需蛋白质纯化。
