Communication between the domains of the DnaK chaperone characterized by single molecule force spectroscopy

DnaK是大肠杆菌Hsp70的伴侣蛋白，由一个n端核苷酸结合域（NBD）和一个c端底物结合域（SBD）组成。NBD结合ATP或ADP，表现出非常弱的ATP酶活性。SBD识别蛋白质底物多肽链中的疏水延伸。DnaK在ATP与NBD的结合和蛋白底物与远端SBD的结合之间表现出负协同性。这种双向变构通信是通过结构域之间的保守连接体介导的。值得注意的是，通信途径的潜在物理原理仍然不为人所知。原子力显微镜（AFM）的最新进展允许以亚皮牛顿分辨率和可变维度测量分子力，这对于变构蛋白来说尤其有趣。使用这些技术，我的主要目标将是阐明：(1)不同状态下DnaK的域间动态；(2)核苷酸与底物结合时变构转变的机制；(3)共伴侣DnaJ和核苷酸交换蛋白GrpE协同作用诱导的DnaK亚结构域的运动。

DnaK is a Hsp70 chaperone of E. coli which consists of an N-terminal nucleotide binding domain (NBD) and a C-terminal substrate binding domain (SBD). NBD binds ATP or ADP and displays very weak ATPase activity. SBD recognizes hydrophobic stretches in the polypeptide chain of protein substrates. DnaK shows negative cooperativity between the binding of ATP to NBD and protein substrates to the remote SBD. This bidirectional allosteric communication is mediated through the conserved linker between the domains. Notably, the underlying physical principles of the communication pathways are still not know. Recent progress in atomic force microscopy (AFM) allows molecular forces to be measured at sub-pico-Newton resolution and at variable dimensions what could be particularly interesting for allosteric proteins. Using these techniques, my major goals will be to elucidate: (1) the interdomain dynamics of DnaK in different states; (2) the mechanics of the allosteric transitions upon nucleotide and substrate binding; and (3) the movements of DnaK subdomains induced by the concerted action of the co-chaperone DnaJ and the nucleotide exchange protein GrpE.