An essential proline in lambda repressor is required for resistance to intracellular proteolysis.
An essential proline in lambda repressor is required for resistance to intracellular proteolysis.
复制标题
λ 阻遏蛋白中必需的脯氨酸是抵抗细胞内蛋白水解作用所必需的。
DOI：
10.1021/bi00485a004
复制
发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Sauer,RT
中科院分区：
文献类型：
--
作者：
Reidhaar-Olson,JF;Parsell,DA;Sauer,RT
生物系，马萨诸塞州理工学院，剑桥，马萨诸塞州02139接收于1990年2月28日;修订的Mandarin pt接收于1990年5月11日摘要：Pro78是X阻遏物DNA结合域中α-螺旋5 N-末端的溶剂暴露残基。随机诱变实验表明，Pro78是必需的[Reidhaar-Olson，JF，& Sauer，R. T. 03 The Dog of the Woman（1990）功能：Genet，（出版中）]。为了研究这个位置对脯氨酸的需求，我们构建了一组10个78位突变蛋白并研究了其性质。所有这些突变体都具有降低的细胞内活性，并且以显著低于野生型的水平表达。脉冲追踪实验表明，突变蛋白在细胞中迅速降解;检测的突变体的半衰期为11-35分钟，而野生型蛋白的半衰期大于10小时。位置78突变体的快速降解不受影响已知大肠杆菌蛋白酶的突变的抑制。Pro78-Ala突变体可以在dnaJ-菌株中过表达并被纯化。该突变体在体外具有完整的DNA结合活性，表明其折叠结构和形成活性二聚体的能力与野生型相似。PA 78突变体（Tm= 48 ℃）的热稳定性低于野生型（Tm= 55 ℃）.双突变体研究表明，这种不稳定性有助于但不是其快速细胞内降解的主要原因，也表明蛋白水解是由含有PA 78取代的蛋白质的变性形式引起的。PA 78突变似乎没有引入细胞蛋白酶的新切割位点，该突变也没有增强体外对蛋白酶如嗜热菌蛋白酶和胰蛋白酶的易感性。该突变确实降低了GuHCl变性实验中的m值，并可能改变变性多肽的性质，使其能够被E.大肠杆菌蛋白酶或辅助降解因子。
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received February 28, 1990; Revised Manuscript Received May 11, 1990 abstract: Pro78 is a solvent-exposed residue at the N-terminal end of a-helix 5 in the DNA binding domain of X repressor. Random mutagenesis experiments have suggested that Pro78 is essential [Reidhaar-Olson, JF, & Sauer, R. T.(1990) Proteins: Struct., Funct., Genet,(in press)]. To investigate the requirement for prolineat this position, we constructed and studied the properties of a set of ten position 78 mutant proteins. All of these mutants have decreased intracellular activities and are expressed at significantly lower levels than wild type. Pulse-chase experiments show that themutant proteins are rapidly degraded in the cell; the mutants examined had half-lives of 11-35 min, whereas the wild-type protein has a half-life of greater than 10 h. The rapid degradation of position 78 mutants is not suppressed by mutationsthat affect known Escherichia coli proteases. The Pro78-» Ala mutant could be overexpressed in a dnaJ~ strain and was purified. This mutant has full DNA binding activity in vitro, suggesting that its foldedstructure and ability to form active dimers are similar to those of wild type. The PA78 mutant (Tm= 48 C) is less thermally stable than wild type (Tm= 55 C). Double-mutant studies show that this instability contributes to but is not the main cause of its rapid intracellular degradation and also suggest that proteolysisproceeds from the denatured forms of proteins containing the PA78 substitution. The PA78 mutation does not appear to introduce a new cleavage site for cellular proteases, nor does the mutation enhance susceptibility to proteases such as thermolysin and trypsin in vitro. The mutation does decrease the m value in GuHCl denaturation experiments and may alter the properties of the denatured polypeptide, allowing it to be specifically recognized by an E. coli protease or auxiliary degradation factor.