热休克蛋白90(Hsp90)家族的伴侣是众所周知的,调节蛋白质稳态的细胞系统的重要组成部分。在真核生物中必不可少,Hsp90也存在于原核生物中,包括古细菌。Hsp90是一种二聚体蛋白,每个单体由三个独立的结构域组成,并经历了大的构象变化,作为其功能循环的一部分。这个循环是由与核苷酸的相互作用驱动的,伴侣蛋白,客户端蛋白质和变构效应由这些和翻译后修饰产生。所有这些都会影响二聚体的打开和关闭的速率和程度以及相对畴取向及其整体刚度。光学镊子,可以获得许多功能上重要的构象变化,因此为研究这种庞大而复杂的分子伴侣提供了独特的工具。这里,我们为不同的Hsp90构建体的设计和实现提供了协议,并提供了光学镊子实验,以解决有关这种重要分子伴侣功能的许多悬而未决的问题。
The heat shock protein 90 (Hsp90) family of chaperones are well-known, highly important components of the cellular systems which regulate protein homeostasis. Essential in eukaryotes, Hsp90s is also found in prokaryotes, including archaea. Hsp90 is a dimeric protein, with each monomer consisting of three separate structural domains, and undergoes large conformational changes as part of its functional cycle. This cycle is driven by interactions with nucleotides, cochaperone proteins, client proteins and allosteric effects enacted by these and by posttranslational modifications. All of these influence the rate and degree of the opening and closing of the dimer as well as the relative domain orientations and its overall rigidity. Optical tweezers, which can access many of these functionally important conformational changes, therefore provide a unique tool for the
study of this large and complex molecular chaperone. Here, we provide protocols for the design and implementation of different Hsp90 constructs and optical tweezers experiments for addressing the many open questions about the function of this important molecular chaperone.