Sather S K, King J
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
J Biol Chem. 1994 Oct 14;269(41):25268-76.
Critical steps in polypeptide chain folding within the bacterial cytoplasm have been difficult to identify. Salmonella cells infected with temperature-sensitive folding mutants of the P22 tailspike protein at restrictive temperature accumulated a metastable folding intermediate with a half-life of 6 min at 39 degrees C. The native trimeric tailspike contains 24 buried cysteines (8/chain) but neither disulfide bonds nor active site cysteines. Eighteen of the 24 cysteines are involved in strong hydrogen bonds (Thomas, G. J., Jr., Becka, R., Sargent, D., Yu, M.-H., and King, J. (1990) Biochemistry 29, 4181-4187). Cyanide and iodoacetamide prevented the folding and association of the restrictive temperature folding intermediate to the native state after shift to permissive temperature. The cytoplasmic folding intermediate was covalently modified by iodoacetamide within infected cells. Chains which had reacted with iodoacetamide were unable to proceed through the folding pathway. Iodoacetamide also reacted with a folding intermediate during the refolding of purified tailspike chains in vitro, inhibiting further folding. No reaction occurred with native tailspike in vivo or in vitro. The target residues in the intermediates were in the carboxyl terminus of the chain and may be a unique set of cysteine residues that are activated during protein folding, but not in the native state.
细菌细胞质中多肽链折叠的关键步骤一直难以确定。在限制温度下感染P22尾刺蛋白温度敏感型折叠突变体的沙门氏菌细胞积累了一种亚稳态折叠中间体，在39℃时半衰期为6分钟。天然三聚体尾刺含有24个埋藏的半胱氨酸（每条链8个），但既没有二硫键也没有活性位点半胱氨酸。24个半胱氨酸中有18个参与强氢键（小托马斯·G·J、贝卡·R、萨金特·D、于美华和金·J（1990年）《生物化学》29卷，4181 - 4187页）。氰化物和碘乙酰胺在转移到允许温度后阻止了限制温度折叠中间体折叠并缔合到天然状态。感染细胞内的碘乙酰胺对细胞质折叠中间体进行了共价修饰。与碘乙酰胺反应的链无法继续通过折叠途径。在体外纯化尾刺链重折叠过程中，碘乙酰胺也与一种折叠中间体发生反应，抑制了进一步折叠。在体内或体外，碘乙酰胺与天然尾刺均无反应。中间体中的目标残基位于链的羧基末端，可能是一组独特的半胱氨酸残基，它们在蛋白质折叠过程中被激活，但在天然状态下未被激活。
