Structure and Mechanism of C1pB from E. coli

描述(申请人提供)：C1pB是大肠杆菌中蛋白质解聚多伴侣系统的成员。它的三维结构和C1pB介导的蛋白质折叠活性的机制目前尚不清楚。C1pB和几个截短的结构已经被过度表达和研究。我们已经获得了野生型C1pB和含有N-末端769个残基的构建体C1pBdeltaC的表达系统。结晶努力得到了ClpBdeltaC的晶体。利用X射线衍射法确定这些晶体的结构是该提案的第一个主要目标。这将通过经典的多重同象取代法或通过酶的硒蛋氨酸形式的多波长反常衍射(MAD)来实现。在这些研究之后，将进行ATP(或其非水解性类似物)晶体结合研究。我们将继续努力使全长ClpB具体化。如果我们成功，它的结构将用C1pBdeltaC模型的分子置换方法来确定。如果结晶不成功，我们将使用同源建模来获得缺失的C-末端部分的结构。Hs1U(也称为C1pY)的结构是可用的，其残基1-287与C1pB的残基551-857有25%的序列同源性。目前还没有与ClpB的N-末端550个氨基酸同源的蛋白质的已知结构。

DESCRIPTION (provided by applicant): C1pB is a member of a protein-disaggregating multi-chaperone system in Escherichia coli. Its three-dimensional structure and the mechanism of protein-folding activity mediated by C1pB is currently unknown. C1pB and several truncated constructs have been overexpressed and studied. We have received the expression system for the wild type C1pB and for a construct containing N-terminal 769 residues (out of 857), C1pBdeltaC. Crystallization efforts yielded crystals of ClpBdeltaC. The determination of the structure of these crystals using X-ray diffraction is the first, major objective of the proposal. It will be achieved either by the classical multiple isomorphous replacent method or by multiwavelength anomalous diffraction (MAD) of the selenomethionine form of the enzyme. These studies will be followed by ATP (or its non-hydrolyzable analogue) crystal binding studies. We will continue our efforts to crystallize the full length ClpB. If we are successful its structure will be determined using the molecular replacement method with the model of C1pBdeltaC. If we are not successful in crystallization, we will use homology modeling to obtain the structure of the missing C-terminal part. The structure of Hs1U (also known as C1pY) is avaliable and its residues 1-287 share 25% sequence identity with residues 551-857 of C1pB. There is no known structure of a protein homologous to the N-terminal 550 amino acids of ClpB.