Sato Katsuko, Li Chan, Salard Isabelle, Thompson Andrew J, Banfield Mark J, Dennison Christopher
Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom.
Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5616-21. doi: 10.1073/pnas.0811324106. Epub 2009 Mar 19.
The C-terminal copper-binding loop in the beta-barrel fold of the cupredoxin azurin has been replaced with a range of sequences containing alanine, glycine, and valine residues to assess the importance of amino acid composition and the length of this region. The introduction of 2 and 4 alanines between the coordinating Cys, His, and Met results in loop structures matching those in naturally occurring proteins with the same loop lengths. A loop with 4 alanines between the Cys and His and 3 between the His and Met ligands has a structure identical to that of the WT protein, whose loop is the same length. Loop structure is dictated by length and not sequence allowing the properties of the main surface patch for interactions with partners, to which the loop is a major contributor, to be optimized. Loops with 2 amino acids between the ligands using glycine, alanine, and valine residues have been compared. An empirical relationship is found between copper site protection by the loop and reduction potential. A loop adorned with 4 methyl groups is sufficient to protect the copper ion, enabling most sequences to adequately perform this task. The mutant with 3 alanine residues between the ligands forms a strand-swapped dimer in the crystal structure, an arrangement that has not, to our knowledge, been seen previously for this family of proteins. Cupredoxins function as redox shuttles and are required to be monomeric; therefore, none have evolved with a metal-binding loop of this length.
在铜蓝蛋白天青蛋白的β桶折叠结构中，其C末端铜结合环已被一系列含有丙氨酸、甘氨酸和缬氨酸残基的序列所取代，以评估氨基酸组成和该区域长度的重要性。在配位的半胱氨酸、组氨酸和甲硫氨酸之间引入2个和4个丙氨酸，会产生与具有相同环长度的天然蛋白质中的环结构相匹配的环结构。在半胱氨酸和组氨酸之间有4个丙氨酸且在组氨酸和甲硫氨酸配体之间有3个丙氨酸的环，其结构与野生型蛋白相同，野生型蛋白的环长度也是如此。环结构由长度而非序列决定，这使得作为与伙伴相互作用主要贡献者的主要表面斑块的性质得以优化。已比较了在配体之间使用甘氨酸、丙氨酸和缬氨酸残基的含2个氨基酸的环。发现环对铜位点的保护与还原电位之间存在经验关系。带有4个甲基修饰的环足以保护铜离子，使大多数序列能够充分完成这项任务。在配体之间有3个丙氨酸残基的突变体在晶体结构中形成了链交换二聚体，据我们所知，这种排列在该蛋白家族中以前从未见过。铜蓝蛋白作为氧化还原穿梭体发挥作用，需要是单体形式；因此，没有一种铜蓝蛋白进化出这种长度的金属结合环。
