Structural and mechanistic studies of Hsp 100 protein

Hsp 100 蛋白的结构和机制研究

• 批准号: 6615749
• 负责人: BINGDONG SHA
• 金额: $ 25.38万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6615749
• 关键词: Escherichia coli; X ray crystallography; adenosine diphosphate; adenosine triphosphate; adenosinetriphosphatase; conformation; electron microscopy; gene mutation; heat shock proteins; peptides; protein binding; protein sequence; protein structure; structural biology

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.

描述（由申请人提供）：该提案的目标是在HSPL 00上进行结构研究，以揭示其用作分子伴侣的机制。大肠杆菌HSPL 00 CLPB最近通过分解非本性多肽来确定为分子伴侣。为了研究Hsploo CIPB分解非本地多肽的机制，我们建议确定CLPB N末端结构域结构。 CIPB N末端结构域已显示与非本性多肽直接相互作用，并在CIPB伴侣函数中起重要作用。我们已经将CLPB的N末端结构域和晶体衍射为I .95a结晶。在CIPB N末端结构域的结构中，我们可以识别一个肽结合的凹槽。因此，我们可以预测由HSPL 00 CIPB结合的肽的最小长度。 CIPB的高亲和肽底物将通过遗传和生物物理方法鉴定。我们将结晶CIPB N末端结构域与其肽底物的复合物。该复合物的晶体结构将提供有关Hsploo CIPB如何识别和结合非本性多肽的基本见解。为了揭示CIPB进行其ATPase活性的机制，我们建议用C-末端片段（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域NBDI和NBD2。我们已经解决了CIPB NBDI的晶体结构，并具有与ATP或ADP复合的结晶CIPB D2C。为了测试模型的Hsploo CIPB伴侣功能的机制，我们将构建两组基于结构的CIPB突变体。一种是在CLPB N末端结构域中突变残基，这些残基严重涉及结合肽。这些突变体将通过肽结合测定和蛋白质折叠测定法测试功能损失。另一组突变体是支持CIPB ATPase活性的建议的“透明”模型。我们将突变残基，以禁用CIPB C末端片段的构象变化。突变体将通过核苷酸结合测定，ATPase活性测定和蛋白质折叠测定来测试该功能。总的来说，该提案涵盖了一项综合研究，该研究揭示了CLPB与非本性多肽相互作用并进行ATP水解以充当分子伴侣的机制。