Reduction potential variations in azurin through secondary coordination sphere phenylalanine incorporations
Reduction potential variations in azurin through secondary coordination sphere phenylalanine incorporations
复制标题
DOI：
10.1016/j.jinorgbio.2010.06.004
复制
发表时间：
2010-10-01
影响因子：
3.9
通讯作者：
Reardon, Nicole J.
中科院分区：
文献类型：
--
作者：
Berry, Steven M.;Baker, Madelyn H.;Reardon, Nicole J.
最近的证据表明，金属结合位点的性质可以通过比初级配位球中的配体更多的配体来调节。我们研究了四个苯丙氨酸残基的小可溶性蓝铜蛋白天青蛋白的二级配位球的纳入。这些残基在天青蛋白中的位置是基于高度疏水的蓝铜蛋白rusticyanin的结构，已知其具有比天青蛋白显著更高的还原电位。使用定点诱变，这些残基在接近铜结合位点突变为大的疏水性苯丙氨酸残基单独和组合。我们还在Phe突变之上添加了Met 121 Leu突变，以构建总共13个变体。我们发现这些蛋白质的UV-可见光吸收和EPR数据变化不大，但是观察到还原电位适度增加，每个Phe残基增加多达30 mV。此外，我们观察到潜在的添加剂的增加。(c)2010年爱思唯尔公司All rights reserved.
Recent evidence has shown that the properties of metal binding sites can be tuned by more than the ligands in the primary coordination sphere. We investigated the incorporation of four phenylalanine residues into the secondary coordination sphere of the small soluble blue copper protein azurin. The locations for placement of these residues in azurin were based on the structure of the highly hydrophobic blue copper protein rusticyanin, which is known to have a significantly higher reduction potential than azurin. Using site-directed mutagenesis, these residues in close proximity to the copper binding site were mutated to large hydrophobic phenylalanine residues individually and in combination. We also added the Met121Leu mutation on top of the Phe mutations to construct a total of 13 variants. We found little change in the UV-visible absorption and EPR data for these proteins, however modest increases in reduction potential were observed with increases by as much as 30 mV per Phe residue. Furthermore, we observed the increases in potential to be additive. (c) 2010 Elsevier Inc. All rights reserved.