Using chemical genetics to explore GroEL function

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，还没有发现严重条件缺乏GroEL的菌株。这样的菌株将使人们能够解决这样的问题：groel缺陷细胞是否继续翻译多肽？在这种条件下，有多少多肽和哪些多肽发生了错误折叠/聚集？包涵体是否形成？是否诱导了其他伴侣？在这里，我们建议通过制造一种化学抑制剂来解决这个问题，这种抑制剂可以穿过大肠杆菌膜，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Asn479和Ile493突变为更小的残基丙氨酸和甘氨酸，以创建一个疏水口袋，可能能够结合一个或多个化学合成的一系列腺嘌呤类似物，这些类似物具有不同位置的大疏水基团。敏化的GroEL突变体和细胞渗透性抑制剂的结合应该允许在体内快速和严重地抑制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。

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.