KINETICS AND MECHANISM OF THE HEAT SHOCK 70 PROTEIN DNAK

本研究的长期目标是阐明细菌伴侣机器促进蛋白质折叠的分子机制，该机器由70 kDa的分子伴侣Dna K、41 kDa的Co伴侣Dna J和23 kDa的核苷酸交换因子GRPE组成。几个特定的目标集中在了解DNAK与用于模拟未折叠底物蛋白质的多肽相互作用的化学。有待阐明的问题是：多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-螺旋盖子如何影响多肽进入和离开DNAK的多肽结合部位；以及β-夹心多肽结合域和α-螺旋盖子之间的盐桥网络如何控制多肽与DNAK的结合。另一个具体目的是剖析辅助伴侣DNAJ和GrpB是如何调节DNAK的活动的。需要阐明的是DNAJ辅助的DNAK从低亲和力到高亲和力结构转换的动力学和机制；DNAJ是否降低了结合到DNAK低亲和力状态的多肽的激活能障碍；以及GRPE辅助从DNAK高亲和力状态释放紧密结合的多肽的动力学和机制。将使用分子生物学的工具来制备DNAK的变体。具体的目标将通过在DNAK变种和伴侣机器上使用停流荧光和表面等离子共振进行各种动力学实验来实现。人们逐渐意识到，随着年龄的增长，一些细胞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，控制未折叠蛋白的数量，减少缺血造成的损伤，甚至促进宿主对癌细胞的攻击；因此，阐明K/J/E伴侣机制可能最终有助于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.