Using chemical genetics to explore GroEL function

利用化学遗传学探索 GroEL 功能

• 批准号: 6739344
• 负责人: Eli Chapman
• 金额: $ 3.59万
• 依托单位: YALE UNIVERSITY
• 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 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6739344
• 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.

细菌细胞质中的伴侣蛋白 GroEL 已被证明有助于多肽链折叠，但迄今为止，还没有严重条件性缺乏 GroEL 的菌株。这样的菌株将允许人们解决以下问题：GroEL 缺陷的细胞是否继续翻译多肽？在这种条件下，有多少以及哪些多肽发生错误折叠/聚集？是否形成包涵体？是否有其他伴侣被诱导？在这里，我们建议通过生产一种化学抑制剂来解决这个问题，该抑制剂将穿过大肠杆菌膜并立即关闭突变致敏的 GroEL 的 ATP 酶，从而阻断伴侣蛋白的作用。基于分子模型研究，我们选择了 ATP 口袋中的两个残基 Asn479 和 Ile493，突变为较小的残基丙氨酸和甘氨酸，以创建一个疏水口袋，该疏水口袋可能能够与一系列化学合成的腺嘌呤类似物结合，这些腺嘌呤类似物在不同位置上附着有大的疏水基团。致敏的 GroEL 突变体和细胞渗透性抑制剂的组合应能够快速且严重地抑制体内 GroEL 功能。 然后，我们将检测表达致敏 GroEL 突变体的大肠杆菌细胞的蛋白质翻译、蛋白质折叠和细胞形态。