Using chemical genetics to explore GroEL function

利用化学遗传学探索 GroEL 功能

• 批准号: 6985881
• 负责人: Eli Chapman
• 金额: $ 0.71万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6985881
• 关键词: adenine analog; adenosinetriphosphatase; bacterial proteins; binding sites; cell morphology; chemical binding; chemical genetics; chemical synthesis; cytoplasm; inhibitor /antagonist; molecular chaperones; protein folding; protein structure function

The chaperonin GroEL in the bacterial cytoplasm has been shown to assist polypeptide chain folding but to date, a strain severely conditionally deficient in GroEL has not been available. Such a strain would allow one to address such questions as: do GroEL-deficient cells continue to translate polypeptides? How many and which polypeptides become misfolded/aggregated under such conditions? Are inclusion bodies formed? Are other chaperones induced? Here we propose to attack this problem by producing a chemical inhibitor that will cross E. coli membranes and immediately shut off the ATPase of a mutationally sensitized GroEL, blocking chaperonin action. Based on molecular modeling studies, we have selected two residues in the ATP pocket, Asn479 and Ile493, to mutate to smaller residues, alanine and glycine, to create a hydrophobic pocket potentially capable of binding one or more of a chemically synthesized series of adenine analogues with large, hydrophobic groups attached at various positions. The combination of the sensitized GroEL mutant and cell permeable inhibitor should allow for the rapid and severe inhibition of GroEL function in vivo. We will then assay protein translation, protein folding, and cell morphology of E. coli cells expressing the sensitized GroEL mutant.

已显示细菌细胞质中的伴侣蛋白GroEL有助于多肽链折叠，但迄今为止，还没有GroEL严重条件性缺陷的菌株。这样的菌株将允许人们解决这样的问题：GroEL缺陷细胞继续翻译多肽吗？在这种条件下，有多少多肽和哪些多肽发生错误折叠/聚集？包涵体形成了吗？其他的伴侣也被诱导了吗？在这里，我们提出了解决这个问题，通过生产一种化学抑制剂，将交叉E。大肠杆菌膜，并立即关闭突变致敏的GroEL的ATP酶，阻断伴侣蛋白的作用。基于分子建模研究，我们选择了ATP口袋中的两个残基Asn 479和Ile 493，突变为更小的残基丙氨酸和甘氨酸，以创建一个疏水口袋，该口袋可能能够结合一个或多个化学合成的腺嘌呤类似物系列，在不同位置连接有大的疏水基团。致敏GroEL突变体和细胞渗透性抑制剂的组合应允许体内GroEL功能的快速和严重抑制。 然后我们将分析蛋白质翻译，蛋白质折叠，和细胞形态的E。表达致敏GroEL突变体的大肠杆菌细胞。