Using chemical genetics to explore GroEL function

利用化学遗传学探索 GroEL 功能

• 批准号: 7008527
• 负责人: Eli Chapman
• 金额: $ 5.04万
• 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/7008527
• 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凹槽已被证明可以有助于多肽链折叠，但迄今为止，尚未有严重有条件地缺乏凹槽的菌株。这样的菌株将使人们能够解决以下问题：凹槽缺陷的细胞是否继续翻译多肽？在这种情况下，有多少和哪些多肽被错误折叠/汇总？包容体形成吗？是否诱发其他伴侣？在这里，我们建议通过产生将越过大肠杆菌膜的化学抑制剂来攻击这个问题，并立即关闭突变敏化的凹槽的ATPase，从而阻止伴侣蛋白的作用。基于分子建模研究，我们在ATP口袋中选择了两个残基ASN479和ILE493，以突变与较小的残基，丙氨酸和甘氨酸，以创建一个潜在的疏水性袋，能够在各个位置附着一个或多个具有化学合成的腺苷类模拟的化学合成系列的腺苷系列。敏化的凹槽突变体和可渗透抑制剂的组合应允许在体内快速和严重抑制凹槽功能。 然后，我们将分析表达敏化gr颗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。