Mitraki A, Danner M, King J, Seckler R
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
J Biol Chem. 1993 Sep 25;268(27):20071-5.
One of the central problems in protein folding is how amino acid sequences within polypeptide chains direct polypeptide chain folding and avoid off-pathway aggregation both in intracellular environments and in the test tube. The tailspike protein of phage P22 is a model system for which genetic analysis has permitted mutational dissection of the role of amino acid positions in the polypeptide chain in directing its in vivo folding. Two classes of mutations that affect intracellular folding and aggregation have been characterized; temperature-sensitive folding (tsf) mutants and second-site suppressors of tsf mutants. Here we report the effects of these mutations on the in vitro refolding and aggregation pathway of the purified proteins. The tsf mutations reduced refolding yields at high temperature and increased aggregation, while second-site suppressors enhanced refolding and inhibited aggregation in the test tube. For both types of mutations, the strength of the effects observed in vitro correlated with their in vivo phenotypes. The results confirm that the mutations act intrinsically on the folding pathway of the tailspike polypeptide and not through accessory proteins.
蛋白质折叠的核心问题之一是多肽链中的氨基酸序列如何指导多肽链折叠，并在细胞内环境和试管中避免错误折叠的聚集。噬菌体P22的尾刺蛋白是一个模型系统，通过遗传分析可以对多肽链中氨基酸位置在指导其体内折叠中的作用进行突变分析。已经鉴定出两类影响细胞内折叠和聚集的突变；温度敏感折叠（tsf）突变体和tsf突变体的第二位点抑制子。在这里，我们报告了这些突变对纯化蛋白体外重折叠和聚集途径的影响。tsf突变降低了高温下的重折叠产率并增加了聚集，而第二位点抑制子在试管中增强了重折叠并抑制了聚集。对于这两种类型的突变，体外观察到的效应强度与其体内表型相关。结果证实，这些突变本质上作用于尾刺多肽的折叠途径，而不是通过辅助蛋白。
