Department of Chemistry and Biochemistry, University of Minnesota Duluth, 1039 University Dr., Duluth, MN 55812, USA.
J Biol Inorg Chem. 2011 Mar;16(3):473-80. doi: 10.1007/s00775-010-0746-7. Epub 2010 Dec 14.
Loop-directed mutagenesis was applied to the blue copper protein azurin to replace its copper binding loop with that from the red copper protein nitrosocyanin. A ten amino acid long loop that provides three of the four copper ligands from nitrosocyanin was incorporated into azurin to make a variant called NC-azurin. The chimeric protein displayed a red color, and UV-vis absorption and EPR spectra that closely resembled those of the loop parent, nitrosocyanin. We added the fourth ligand from nitrosocyanin into NC-azurin, a carboxylate-containing amino acid, but the proteins had altered stability and spectroscopic properties that did not resemble those of either parent copper protein. The loop alone, however, was enough to impart red copper site characteristics to the NC-azurin protein. Finally, the reduction potential of the variant was found to be between the reduction potentials of the parent proteins and about 50 mV below that of wild-type azurin.
采用环定向诱变的方法，将蓝色铜蛋白天青蛋白的铜结合环替换为来自红色铜蛋白亚硝酰铜的环。将一个由十个氨基酸组成的长环插入天青蛋白中，该环提供了来自亚硝酰铜的四个铜配体中的三个，从而得到一个名为 NC-天青蛋白的变体。该嵌合蛋白呈现红色，并且其紫外可见吸收和电子顺磁共振光谱与环亲本亚硝酰铜非常相似。我们将亚硝酰铜的第四个配体，即含有羧基的氨基酸，添加到 NC-天青蛋白中，但这些蛋白的稳定性和光谱性质发生了改变，与任何亲本铜蛋白都不相似。然而，环本身足以赋予 NC-天青蛋白红色铜位的特征。最后，发现变体的还原电位介于亲本蛋白的还原电位之间，比野生型天青蛋白的还原电位低约 50 mV。
