CHAPERONIN AIDED PROTEIN FOLDING OF RHODANESE

伴侣蛋白辅助罗丹酶的蛋白质折叠

• 批准号: 6385330
• 负责人: PAUL M HOROWITZ
• 金额: $ 30.26万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6385330
• 关键词: adenosine triphosphate; binding sites; chemical binding; chemical kinetics; conformation; crosslink; detergents; fluorescent dye /probe; hydrolysis; hydropathy; intermolecular interaction; ions; molecular chaperones; mutant; nucleotide metabolism; nucleotides; protein binding; protein folding; protein structure function; synthetic peptide; thiosulfate sulfurtransferase

We seek to understand the control of protein folding. Cell stress proteins, like the chaperonin GroEL, bind and protect partly folded proteins and prevent misfolding. Controlled release then leads to native conformations. Chaperonins respond to disease, and they are used diagnostically, e.g. for breast cancer. We seek molecular details of GroEL interactions with the enzyme rhodanese-an ideal substrate whose structure and folding are well documented. What general interactions allow GroEL to bind diverse partially-folded but not native proteins? We speculate that the unusual macromolecular recognition displayed by GroEL involves induction of hydrophobic exposure by ionic interactions. We will use functional and biophysical methods to study the complexes of GroEL, its monomers and apical domains that can be formed with rhodanese and the co-chaperonin, GroES. We stress techniques that respond to dynamics and conformation changes in ways that complement static, high resolution methods. The specific Aims are designed to extent ongoing research and test four hypotheses: I. Chaperonin function involves the interplay of GroEL inter-monomer interactions, intra-monomer flexibility, ATP hydrolysis, and dynamic binding of ions, proteins and GroES. Fluorescence and tritium exchanges will monitor flexibility; and hydrodynamic methods will monitor size and quaternary structure. Exchange of GroES and ADP from complex will be compared to test for rate limiting GroEL conformational changes. II. Hydrophobic surfaces on GroEL provide the general interaction for protein binding, and they can be induced and modulated by nucleotides, ions and amphiphiles including proteins and peptides. Fluorescent probes will be used to compare hydrophobic exposure on native or mutant GroEL, its monomers and isolated apical domains. Probes will be used alone, coupled to proteins or photoincorporated into relevant sites. III. Specific interacting regions on target proteins and on GroEL can be identified. We will use cross linking and bisANS photoincorporation to identify binding sites within complexes formed between GroEL and rhodanese mutants or synthetic peptides representing stabilized helices. IV. The conformation stabilities and structures of intermediates formed by partially folded target proteins influence interactions with GroEL. We will study: a) GroEL binding of rhodanese mutants with different stabilities; and b) heterogeneity of bound intermediates.

我们试图了解蛋白质折叠的控制。细胞应激 蛋白质，例如伴侣蛋白凹槽，结合并保护部分折叠 蛋白质并防止错误折叠。受控释放然后导致本地 构象。伴侣蛋白对疾病有反应，它们被使用 诊断，例如用于乳腺癌。我们寻求分子细节 凹槽与罗达尼斯酶的相互作用 - 理想底物 结构和折叠有充分的记录。什么一般互动 允许凹槽结合各种部分折叠但不折叠天然蛋白质？我们 推测凹槽显示的异常大分子识别 涉及通过离子相互作用诱导疏水性暴露。我们 将使用功能和生物物理方法来研究 凹槽，它的单体和顶端域，可以用罗丹人形成 和共伴侣蛋白，凹槽。我们强调响应的技术 动态和构象以补充静态，高的方式变化 解决方法。 具体目标的设计在进行的研究和测试四个 假设： I.伴侣蛋白的功能涉及凹槽间间隙的相互作用 相互作用，理学内柔性，ATP水解和动态 离子，蛋白质和凹槽的结合。荧光和tri频交换 将监视灵活性；和流体动力学方法将监视尺寸和 第四纪结构。与复合物的凹槽和ADP交换将是 与测试速率限制凹槽构象变化相比。 ii。凹槽上的疏水表面提供了一般相互作用 蛋白质结合，可以通过核苷酸诱导和调节， 离子和两亲物，包括蛋白质和肽。荧光探针 将用于比较天然或突变凹槽上的疏水暴露， 它的单体和孤立的顶端域。探针将单独使用， 耦合到蛋白质或光电融合到相关位点。 iii。靶蛋白和凹槽上的特定相互作用区域可以是 确定。我们将使用交叉链接和Bisans photoincorporation 识别凹槽和在凹槽和 罗丹尼突变体或代表稳定螺旋的合成肽。 iv。形成中间体的构象稳定性和结构 通过部分折叠的靶蛋白会影响与凹槽的相互作用。 我们将研究：a）具有不同的罗达尼突变体的凹槽结合 稳定； b）结合中间体的异质性。