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

DnaK是E. coli中，该基因由N端核苷酸结合结构域（NBD）和C端底物结合结构域（SBD）组成。NBD结合ATP或ADP，并显示非常弱的ATP酶活性。SBD识别蛋白质底物的多肽链中的疏水延伸。DnaK在ATP与NBD的结合和蛋白质底物与远端SBD的结合之间显示负协同性。这种双向变构通信通过结构域之间的保守接头介导。值得注意的是，通信路径的基本物理原理仍然不知道。原子力显微镜（AFM）的最新进展允许在亚皮牛顿分辨率和可变尺寸下测量分子力，这对于变构蛋白质可能特别有趣。利用这些技术，我的主要目标将是阐明：（1）在不同状态下的DnaK的域间动力学;（2）在核苷酸和底物结合时的变构转换的机制;和（3）由辅伴侣DnaJ和核苷酸交换蛋白GrpE的协同作用诱导的DnaK亚域的运动。

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.