Constraints on amino acid substitutions in the N-terminal helix of cytochrome c explored by random mutagenesis.
Constraints on amino acid substitutions in the N-terminal helix of cytochrome c explored by random mutagenesis.
复制标题
通过随机诱变探索细胞色素 c N 端螺旋中氨基酸取代的限制。
DOI：
10.1021/bi00099a028
复制
发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Pielak,GJ
中科院分区：
文献类型：
--
作者：
Auld,DS;Pielak,GJ
Douglas S. Auld和Gary J. Pielak**北卡罗来纳大学教堂山分校化学系，北卡罗莱纳州教堂山27599-3290,1991年2月5日；摘要：n端和c端螺旋的相互作用是细胞色素c家族的一个标志。对酿酒酵母iso1 -cytochrome c编码C102T蛋白变异的基因进行了寡脱氧核糖核苷酸定向随机诱变，建立了n端螺旋进化不变残基Gly-6和ph -10的突变文库。将该文库（包含在低拷贝数酵母穿梭噬菌体上）转化为缺乏功能性细胞色素c的酵母菌株，然后选择细胞色素c的功能，发现400个可能的氨基酸取代中有4-10%与功能相容。从显示功能表型的转化体中分离的噬菌体的DNA序列分析阐明了对稳定螺旋界面的要求。碱基残基在6号或10号位置是不耐受的。氨基酸在位置6处有广泛的体积限制。在ph -10上观察到与功能相容的氨基酸取代表明疏水效应酮足以促进螺旋缔合。有严格的限制，限制了与功能一致的组合，但观察到的功能一致的组合的数量例证了蛋白质的可塑性。螺旋的E对是蛋白质三级结构最基本的类型之一。细胞色素c是一个家族。这项工作得到了NIH FIRST奖（GM42501）和石油研究基金会（21597-G）的资助。给作者写信。在进化上保守的-螺旋蛋白中，n端和-端螺旋的配对存在于所有的细胞色素c中（Matthews, 1985）。对来自酿酒酵母（Louie & Brayer, 1990）的iso1 -细胞色素c晶体结构的检查表明，螺旋轴倾斜约为90度，它们的相互作用涉及到包装
Douglas S. Auld and Gary J. Pielak** Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290 Received February 5, 1991; Revised Manuscript Received June 19, 1991 abstract: The interaction of the N-and C-terminal helices is a hallmark of the cytochrome c family. Oligodeoxyribonucleotide-directed random mutagenesis within the gene encodingthe C102T protein variant of Saccharomyces cerevisiae iso-1-cytochrome c was used to generate a library of mutations at the evolutionary invariant residues Gly-6 and Phe-10 in the N-terminal helix. Transformation of this library (contained on a low-copy-number yeast shuttle phagemid) into a yeast strain lacking a functional cytochrome c, followed by selection for cytochrome c function, reveals that 4-10% of the 400 possible amino acid substitutions are compatible with function. DNA sequence analysis of phagemids isolated from transformants exhibiting the functional phenotype elucidates the requirements for a stable helical interface. Basic residues are not tolerated at position 6 or 10. There is a broad volume constraintfor amino acids at position 6. The amino acid substitutions observed to be compatible with function at Phe-10 show that the hydrophobic effectalone is sufficient to promote helical association. There are severe constraints that limit the combinations consistent with function, but the number of functionally consistent combinations observed exemplifies the plasticity of proteins. e pairing of «-helices isone of the most fundamental types of protein tertiary structure. The cytochromes c are a family tThis work was supported by an NIH FIRST Award (GM42501) and by a grant from the Petroleum Research Foundation (21597-G).* Address correspondence to this author. of evolutionarily conserved «-helical proteins, and the pairing of the N-and¿-terminal helices is foundin all cytochromes c (Matthews, 1985). Examination of the crystal structure of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae (Louie & Brayer, 1990) shows that the helical axes are inclined at approximately 90 and their interaction involves the packing