KINETICS AND MECHANISM OF THE HEAT SHOCK 70 PROTEIN DNAK

本研究的长期目标是阐明由70-kDa分子伴侣DnaK、41-kDa共伴侣DnaJ和23-kDa核苷酸交换因子GrpE组成的细菌伴侣机器促进蛋白质折叠的分子机制。几个具体的目标集中在理解DnaK与肽的相互作用的化学，肽用于模拟未折叠的底物蛋白。要阐明的是肽上的总电荷如何影响其与DnaK的低亲和力和高亲和力状态的反应性; α-螺旋盖如何影响肽进出DnaK的多肽结合位点;以及β-夹心多肽结合结构域和α-螺旋盖之间的盐桥网络如何控制肽与DnaK的结合。另一个具体的目的是剖析共伴侣DnaJ和GrpB如何调节DnaK的活性。待阐明的是DnaK中DnaJ辅助的低亲和力到高亲和力结构转换的动力学和机制; DnaJ是否降低肽结合到DnaK的低亲和力状态的活化能屏障;以及GrpE辅助的紧密结合的多肽从DnaK的高亲和力状态释放的动力学和机制。将使用分子生物学工具制备DnaK的变体。具体目标将通过使用停流荧光和表面等离子体共振对DnaK变体和伴侣机器进行各种动力学实验来实现。人们逐渐意识到，随着一些细胞的衰老，它们可能会积累错误折叠的蛋白质，这些错误折叠的蛋白质与淀粉样变性等疾病有关，这些疾病可能会影响各种器官。真核细胞表达DnaK和DnaJ同源物控制未折叠蛋白的量，减少缺血损伤，甚至促进宿主对癌细胞的攻击;因此，阐明K/J/E伴侣机器的机制可能最终会深入了解未折叠蛋白在人类疾病中的作用。

The long-term objective of this research is to elucidate the molecular mechanism by which the bacterial chaperone machine, composed of the 70-kDa molecular chaperone DnaK, the 41-kDa Co- chaperone DnaJ, and the 23-kDa nucleotide exchange factor GrpE, promotes protein folding. Several specific aims focus on understanding the chemistry of the interactions of DnaK with peptides, which serve to mimic unfolded substrate proteins. To be elucidated are how the total charge on a peptide affects its reactivity with both the low-affinity and high-affinity states of DnaK; how the alpha-helical lid affects the entry and exit of peptides into and out of the polypeptide-binding site of DnaK; and how a network of salt bridges between the beta-sandwich polypeptide-binding domain and the alpha-helical lid controls peptide binding to DnaK. Another specific aim is to dissect how the co-chaperones DnaJ and GrpB modulate the activity of DnaK. To be elucidated are the kinetics and mechanism of the DnaJ-assisted low- to-high affinity structural switch in DnaK; whether DnaJ lowers the activation energy barrier to peptide binding to the low-affinity state of DnaK; and the kinetics and mechanism of GrpE-assisted release of tightly bound polypeptides from the high-affinity state of DnaK. Variants of DnaK will be prepared using the tools of molecular biology. The specific aims will be achieved by conducting a variety of kinetic experiments both on the DnaK variants and the chaperone machine using stopped-flow fluorescence and surface plasmon resonance. There is an emerging awareness that as some cells age they may accumulate misfolded proteins, and that these misfolded proteins are linked to diseases such as amyloidosis, which can affect various organs. The expression of DnaK and DnaJ homologs by eukaryotic cells controls the amounts of unfolded proteins, reduces damage due to ischemia and even promotes the host attack against cancer cells; thus, elucidating the mechanism of the K/J/E chaperone machine may ultimately yield insights into the role of unfolded proteins in human diseases.