Auld D S, Pielak G J
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
Biochemistry. 1991 Sep 3;30(35):8684-90. doi: 10.1021/bi00099a028.
The interaction of the N- and C-terminal helices is a hallmark of the cytochrome c family. Oligodeoxyribonucleotide-directed random mutagenesis within the gene encoding the 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 constraint for amino acids at position 6. The amino acid substitutions observed to be compatible with function at Phe-10 show that the hydrophobic effect alone 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.
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。利用寡聚脱氧核糖核苷酸定向随机诱变技术，对酿酒酵母同工酶 1 - 细胞色素 c 的 C102T 蛋白变体编码基因进行诱变，在 N 端螺旋中进化上保守的 Gly - 6 和 Phe - 10 残基处产生一系列突变。将该文库（存在于低拷贝数酵母穿梭噬菌粒上）转化到缺乏功能性细胞色素 c 的酵母菌株中，然后筛选具有细胞色素 c 功能的菌株，结果表明 400 种可能的氨基酸替换中有 4% - 10%与功能兼容。对从表现出功能表型的转化体中分离出的噬菌粒进行 DNA 序列分析，阐明了稳定螺旋界面的要求。在位置 6 或 10 处不允许存在碱性残基。位置 6 处的氨基酸存在广泛的体积限制。在 Phe - 10 处观察到与功能兼容的氨基酸替换表明，仅疏水作用就足以促进螺旋缔合。存在严重的限制，限制了与功能一致的组合，但观察到的功能一致的组合数量体现了蛋白质的可塑性。
