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Stabilized Human Cystatin C Variant L47C/G69C Is a Better Reporter Than the Wild-Type Inhibitor for Characterizing the Thermodynamics of Binding to Cysteine Proteases.
The Protein Journal ( IF 1.9 ) Pub Date : 2019-05-22 , DOI: 10.1007/s10930-019-09839-2
David O Tovar-Anaya 1 , L Irais Vera-Robles 1 , M Teresa Vieyra-Eusebio 1 , Ponciano García-Gutiérrez 1 , Francisco Reyes-Espinosa 1 , Andrés Hernández-Arana 1 , J Alfonso Arroyo-Reyna 1 , Rafael A Zubillaga 1
Affiliation  

Human cystatin C (HCC) binds and inhibits all types of cysteine proteases from the papain family, including cathepsins (a group of enzymes that participate in a variety of physiological processes), which are some of its natural targets. The affinities of diverse proteases for HCC, expressed as equilibrium binding constants (Kb), range from 106 to 1014 M−1. Isothermal titration calorimetry (ITC) is one of the most useful techniques to characterize the thermodynamics of molecular associations, making it possible to dissect the binding free energy into its enthalpic and entropic components. This information, together with the structural changes that occur during the different associations, could enable better understanding of the molecular basis of affinity. Notwithstanding the high sensitivity of modern calorimeters, ITC requires protein concentrations in at least the 10–100 μM range to obtain reliable data, and it is known that HCC forms oligomers in this concentration range. We present herein a comparative study of the structural, thermal stability, and oligomerization properties of HCC and its stabilized variant (sHCC) L47C/G69C (which possesses an additional disulfide bridge) as well as their binding thermodynamics to the protease chymopapain, analyzed by ITC. The results show that, because sHCC remains monomeric, it is a better reporter than wild-type HCC to characterize the thermodynamics of binding to cysteine proteases.

中文翻译:

稳定的人半胱氨酸蛋白酶抑制剂C变体L47C / G69C比野生型抑制剂更好地报道了与半胱氨酸蛋白酶结合的热力学特性。

人胱抑素C(HCC)结合并抑制木瓜蛋白酶家族的所有类型的半胱氨酸蛋白酶,包括组织蛋白酶(一种参与多种生理过程的酶),这是其某些天然靶标。各种蛋白酶对HCC的亲和力,以平衡结合常数(K b)表示,范围为10 6至10 14  M -1。等温滴定热法(ITC)是表征分子缔合热力学的最有用的技术之一,使将结合自由能分解成其焓和熵组分成为可能。该信息,以及在不同关联过程中发生的结构变化,可以使人们更好地了解亲和力的分子基础。尽管现代量热仪具有很高的灵敏度,但ITC要求蛋白质浓度至少在10-100μM范围内才能获得可靠的数据,并且众所周知,HCC在该浓度范围内会形成寡聚物。我们在此提供结构,热稳定性,ITC分析了HCC及其稳定变异体(sHCC)L47C / G69C(具有一个额外的二硫键)的低聚特性以及它们与蛋白酶糜蛋白酶的结合热力学。结果表明,由于sHCC仍然是单体,因此与野生型HCC相比,它更能报告与半胱氨酸蛋白酶结合的热力学特征。
更新日期:2019-05-22
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