Structural and mechanistic studies of Hsp 100 protein

描述（由申请人提供）：本提案的目标是开展hspl00的结构研究，以揭示其作为分子伴侣的机制。大肠杆菌Hspl 00 ClpB最近被鉴定为通过分解非天然多肽作为分子伴侣。为了研究HsplOO CIpB分解非天然多肽的机制，我们提出确定ClpB n端结构域结构。CIpB n端结构域已被证明与非天然多肽直接相互作用，并在CIpB伴侣功能中发挥重要作用。我们对ClpB的n端畴进行了结晶，晶体的x射线衍射值为1.95 a。在CIpB n端结构域的结构中，我们可以发现一个肽结合槽。因此，我们可以预测hspl00cipb结合肽的最小长度。CIpB的高亲和力肽底物将通过遗传和生物物理方法进行鉴定。我们将使CIpB n端结构域的复合物与其肽底物结晶。该复合物的晶体结构将提供HsplOO CIpB如何识别和结合非天然多肽的基本见解。为了揭示CIpB进行atp酶活性的机制，我们建议用c端片段（D2C）确定CIpB核苷酸结合结构域2的晶体结构。CIpB包含两个核苷酸结合结构域NBDI和NBD2。我们解出了CIpB NBDI的晶体结构，并将CIpB D2C与ATP或ADP络合结晶。为了测试HsplOO CIpB伴侣功能机制的模型，我们将构建两组基于结构的CIpB突变体。一种是突变ClpB n端结构域内的残基，这些残基与结合肽密切相关。这些突变体将通过肽结合试验和蛋白质折叠试验来检测功能的丧失。另一组突变体支持CIpB atp酶活性的“跷跷板”模型。我们将突变残基以禁用CIpB c端片段的构象变化。突变体将通过核苷酸结合试验、atp酶活性试验和蛋白质折叠试验进行功能测试。总的来说，本提案涵盖了一项全面的研究，揭示了ClpB与非天然多肽相互作用并进行ATP水解以作为分子伴侣的机制。

DESCRIPTION (provided by applicant): The goals of this proposal are to carry out structural studies on Hspl 00 to uncover the mechanisms by which it functions as a molecular chaperone. E. coli Hspl 00 ClpB was recently identified to act as a molecular chaperone by disaggregating non-native polypeptides. To investigate the mechanisms for HsplOO CIpB to disaggregate non-native polypeptides, we propose to determine ClpB N-terminal domain structure. The CIpB N-terminal domain has been shown to interact directly with non-native polypeptides and play essential roles in CIpB chaperone functions. We have crystallized the N-terminal domain of ClpB and the crystals diffracted X-ray to I .95A. In the structure of CIpB N-terminal domain, we may identify a peptide-binding groove. Therefore, we could predict the minimal length of peptides bound by Hspl 00 CIpB. The high affinity peptide substrates of CIpB will be identified by genetic and biophysical approaches. We will crystallize the complex of CIpB N-terminal domain with its peptide substrate. The crystal structure of the complex will provide fundamental insights on how HsplOO CIpB recognizes and binds the non-native polypeptides. To reveal the mechanisms for CIpB to carry out its ATPase activities, we propose to determine the crystal structure of CIpB nucleotide-binding domain 2 with the C-terminal fragment (D2C). CIpB contains two nucleotide-binding domains NBDI and NBD2. We have solved the crystal structure of CIpB NBDI and have crystallized CIpB D2C complexed with ATP or ADP. To test the models for the mechanisms for HsplOO CIpB chaperone functions, we will construct two sets of structure-based CIpB mutants. One is to mutate residues within ClpB N-terminal domain that are critically involved in binding peptides. These mutants will be tested for loss of functions by peptide binding assays and protein folding assays. The other set of mutants is to support the proposed "See-Saw" model for CIpB ATPase activity. We will mutate residues to disable the conformational changes of the CIpB C-terminal fragment. The mutants will be tested for functions by nucleotide binding assays, ATPase activity assays and protein folding assays. Collectively, this proposal covers a comprehensive study that reveals the mechanisms by which ClpB interacts with non-native polypeptides and perform ATP hydrolysis to function as a molecular chaperone.