Bhuwan Manish, Suragani Madhuri, Ehtesham Nasreen Z, Hasnain Seyed E
Molecular Infection and Functional Biology Laboratory, Kusuma School of Biological Sciences, Indian Institute of Technology, New Delhi, India.
Department of Biochemistry, School of Life Sciences, University of Hyderabad, Professor C.R. Rao Road, Hyderabad, India.
Bio Protoc. 2017 Jan 20;7(2):e2107. doi: 10.21769/BioProtoc.2107.
The ability to stabilize other proteins against thermal aggregation is one of the major characteristics of chaperone proteins. Molecular chaperones bind to nonnative conformations of proteins. Folding of the substrate is triggered by a dynamic association and dissociation cycles which keep the substrate protein on track of the folding pathway (Figure 1). Usually molecular chaperones exhibit differential affinities with different conformations of the substrate. With the exception of the sHsp family of molecular chaperones, the shift from a high-affinity binding state to the low-affinity release state is triggered by ATP binding and hydrolysis (Haselback and Buchner, 2015). Aggregation prevention assay is a simple, yet definitive assay to determine the chaperone activity of heat labile proteins such as Maltodextrin glucosidase (MalZ), Citrate Synthase (CS) and I. This is based on the premise that proteins with chaperone like activity should prevent protein substrates (MalZ, CS and I) from thermal aggregation. Here, we describe a detailed protocol for aggregation prevention assay using two different chaperone proteins, resistin and MoxR1, identified from our lab. Resistin, a human protein (hRes) and MoxR1 a protein were analysed for their effect on prevention of MalZ/Citrate Synthase (CS)/I aggregation. Figure 1.Mechanism of action of molecular chaperones. Citrate synthase folds via increasingly structured intermediates (I, I) from the unfolded state (U) to the folded state (N). Under heat shock conditions, this process is reversed.
稳定其他蛋白质以防止热聚集的能力是伴侣蛋白的主要特性之一。分子伴侣与蛋白质的非天然构象结合。底物的折叠由动态的结合和解离循环触发，这些循环使底物蛋白保持在折叠途径上（图1）。通常，分子伴侣对底物的不同构象表现出不同的亲和力。除了小分子热休克蛋白（sHsp）家族的分子伴侣外，从高亲和力结合状态到低亲和力释放状态的转变是由ATP结合和水解触发的（哈塞尔巴克和布赫纳，2015年）。聚集预防试验是一种简单但确定的试验，用于测定热不稳定蛋白如麦芽糖糊精葡萄糖苷酶（MalZ）、柠檬酸合酶（CS）和I的伴侣活性。这是基于这样一个前提，即具有类似伴侣活性的蛋白质应该防止蛋白质底物（MalZ、CS和I）发生热聚集。在这里，我们描述了一种详细的聚集预防试验方案，该方案使用了从我们实验室鉴定出的两种不同的伴侣蛋白，抵抗素和MoxR1。对人源蛋白抵抗素（hRes）和蛋白MoxR1对MalZ/柠檬酸合酶（CS）/I聚集预防的影响进行了分析。图1.分子伴侣的作用机制。柠檬酸合酶从未折叠状态（U）通过结构逐渐增加的中间体（I、I）折叠到折叠状态（N）。在热休克条件下，这个过程是相反的。
