Ciglic M I, Jackson P J, Raillard S A, Haugg M, Jermann T M, Opitz J G, Trabesinger-Rüf N, Benner S A
Department of Chemistry, University of Florida, Gainesville 32611-7200, USA.
Biochemistry. 1998 Mar 24;37(12):4008-22. doi: 10.1021/bi972203e.
To enable application of postgenomic evolutionary approaches to understand the divergence of behavior and function in ribonucleases (RNases), the impact of divergent sequence on the divergence of tertiary and quaternary structure is analyzed in bovine pancreatic and seminal ribonucleases, which differ by 23 amino acids. In a crystal, seminal RNase is a homodimer joined by two "antiparallel" intersubunit disulfide bonds between Cys-31 from one subunit and Cys-32' from the other and having composite active sites arising from the "swap" of residues 1-20 from each subunit. Specialized Edman degradation techniques have completed the structural characterization of the dimer in solution, new cross-linking methods have been developed to assess the swap, and sequence determinants of quaternary structure have been explored by protein engineering using the reconstructed evolutionary history of the protein family as a guide. A single Cys at either position 32 (the first to be introduced during the divergent evolution of the family) or 31 converts monomeric RNase A into a dimer. Even with an additional Phe at position 31, another residue introduced early in the seminal lineage, swap is minimal. A hydrophobic contact formed by Leu-28, however, also introduced early in the seminal lineage, increases the amount of "antiparallel" connectivity of the two subunits and facilitates swapping of residues 1-20. Efficient swapping requires addition of a Pro at position 19, a residue also introduced early in the divergent evolution of the seminal RNase gene. Additional cysteines required for dimer formation are found to slow refolding of the protein through formation of incorrect disulfide bonds, suggesting a paradox in the biosynthesis of the protein. Further studies showed that the dimeric form of seminal RNase known in the crystal is not the only form in vivo, where a substantial amount of heterodimer is known. These data complete the acquisition of the background needed to understand the evolution of new structure, behavior, and function in the seminal RNase family of proteins.
为了应用后基因组进化方法来理解核糖核酸酶（RNase）行为和功能的差异，我们分析了牛胰核糖核酸酶和精浆核糖核酸酶中序列差异对三级和四级结构差异的影响，这两种酶有23个氨基酸的差异。在晶体中，精浆核糖核酸酶是一种同型二聚体，由一个亚基的Cys-31与另一个亚基的Cys-32'之间的两个“反平行”亚基间二硫键连接而成，并且具有由每个亚基的1-20位残基“交换”产生的复合活性位点。专门的埃德曼降解技术已完成了溶液中二聚体的结构表征，已开发出新的交联方法来评估这种交换，并且以蛋白质家族的重建进化史为指导，通过蛋白质工程探索了四级结构的序列决定因素。在位置32（该家族分化进化过程中最早引入的位点）或31处的单个半胱氨酸可将单体核糖核酸酶A转化为二聚体。即使在位置31处还有一个额外的苯丙氨酸（也是精浆谱系中早期引入的另一个残基），交换也很少。然而，由亮氨酸-28形成的疏水接触（同样在精浆谱系中早期引入）增加了两个亚基的“反平行”连接量，并促进了1-20位残基的交换。高效交换需要在位置19处添加一个脯氨酸（也是在精浆核糖核酸酶基因分化进化早期引入的残基）。发现二聚体形成所需的额外半胱氨酸会通过形成不正确的二硫键而减慢蛋白质的重折叠，这表明该蛋白质的生物合成存在矛盾。进一步的研究表明，晶体中已知的精浆核糖核酸酶二聚体形式并非体内唯一的形式，已知体内存在大量异二聚体。这些数据完善了理解精浆核糖核酸酶蛋白家族新结构、行为和功能进化所需的背景知识。
