Fontana Angelo, de Laureto Patrizia Polverino, Spolaore Barbara, Frare Erica
CRIBI Biotechnology Centre, University of Padua, Padua, Italy.
Methods Mol Biol. 2012;896:297-318. doi: 10.1007/978-1-4614-3704-8_20.
Limited proteolysis experiments can be successfully used to detect sites of disorder in otherwise folded globular proteins. The approach relies on the fact that the proteolysis of a polypeptide substrate requires its binding in an extended conformation at the protease's active site and thus an enhanced backbone flexibility or local unfolding of the site of proteolytic attack. A striking correlation was found between sites of limited proteolysis and sites of enhanced chain flexibility of the polypeptide chain, this last evaluated by the crystallographically determined B-factor. In numerous cases, it has been shown that limited proteolysis occurs at chain regions characterized by missing electron density and thus being disordered. Therefore, limited proteolysis is a simple and reliable experimental technique that can detect sites of disorder in proteins, thus complementing the results that can be obtained by the use of other physicochemical and computational approaches.
有限蛋白酶解实验可成功用于检测原本折叠的球状蛋白质中的无序位点。该方法基于这样一个事实，即多肽底物的蛋白酶解需要其以伸展构象结合在蛋白酶的活性位点，因此蛋白水解攻击位点的主链灵活性增强或局部解折叠。在有限蛋白酶解位点与多肽链链段灵活性增强位点之间发现了显著的相关性，后者通过晶体学测定的B因子进行评估。在许多情况下，已表明有限蛋白酶解发生在以电子密度缺失为特征因而无序的链区域。因此，有限蛋白酶解是一种简单可靠的实验技术，可检测蛋白质中的无序位点，从而补充通过使用其他物理化学和计算方法所能获得的结果。
