Buried hydrophobic side-chains essential for the folding of the parallel β-helix domains of the P22 tailspike
Buried hydrophobic side-chains essential for the folding of the parallel β-helix domains of the P22 tailspike
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DOI：
10.1110/ps.04676704
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发表时间：
2004-09-01
期刊：
影响因子：
8
通讯作者：
King, J
中科院分区：
文献类型：
--
作者：
Betts, S;Haase-Pettingell, C;King, J
多肽平行的β-螺旋的过程中的β-链和转弯代表了拓扑上最简单的β-折叠之一。噬菌体P22的尾钉粘附素的三个亚基每个都包含13个平行的β-螺旋，紧随其后的是一个交错的三链β-螺旋。长堆叠的疏水残基支配着这两个β-螺旋结构域的细长的埋藏核心，并延伸到连续的三个β-棱柱域的核心。为了测试这些侧链堆叠是否代表将链驱动到正确折叠中的必要残基，掩埋核心内的三个堆叠的苯丙氨酸残基中的每一个都被较小体积的氨基酸取代。用丙氨酸取代苯丙氨酸的突变链在细胞内折叠有缺陷。无论折叠温度如何，链都以聚集的包裹体状态聚集。这些严重的折叠缺陷表明，堆积的苯丙氨酸残基对于正确的平行β-螺旋折叠是必不可少的。用缬氨酸或亮氨酸取代相同的苯丙氨酸残基也会损害体内的折叠，但程度较轻。突变体也被构建在第二个埋藏的堆栈中，该堆栈延伸到蛋白质的相互缠绕的三链β-螺旋和连续的β-棱柱区。这些突变体在链折叠或组装的后期阶段表现出严重的缺陷，以错误折叠但可溶的多聚体的形式积累。结果表明，埋在地下的疏水堆栈的形成对于Tail Spike蛋白中平行的β-螺旋、三链的β-螺旋和β-棱镜结构域的正确折叠至关重要。
The processive beta-strands and turns of a polypeptide parallel beta-helix represent one of the topologically simplest beta-sheet folds. The three subunits of the tailspike adhesin of phage P22 each contain 13 rungs of a parallel beta-helix followed by an interdigitated section of triple-stranded beta-helix. Long stacks of hydrophobic residues dominate the elongated buried core of these two beta-helix domains and extend into the core of the contiguous triple beta-prism domain. To test whether these side-chain stacks represent essential residues for driving the chain into the correct fold, each of three stacked phenylalanine residues within the buried core were substituted with less bulky amino acids. The mutant chains with alanine in place of phenylalanine were defective in intracellular folding. The chains accumulated exclusively in the aggregated inclusion body state regardless of temperature of folding. These severe folding defects indicate that the stacked phenylalanine residues are essential for correct parallel beta-helix folding. Replacement of the same phenylalanine residues with valine or leucine also impaired folding in vivo, but with less severity. Mutants were also constructed in a second buried stack that extends into the intertwined triple-stranded beta-helix and contiguous beta-prism regions of the protein. These mutants exhibited severe defects in later stages of chain folding or assembly, accumulating as misfolded but soluble multimeric species. The results indicate that the formation of the buried hydrophobic stacks is critical for the correct folding of the parallel beta-helix, triple-stranded beta-helix, and beta-prism domains in the tailspike protein.