Kim H W, Shen T J, Sun D P, Ho N T, Madrid M, Ho C
Department of Biological Sciences, Carnegie Mellon University, PA, U.S.A.
J Mol Biol. 1995 May 12;248(4):867-82. doi: 10.1006/jmbi.1995.0267.
Using our Escherichia coli expression plasmid (pHE2) in which synthetic human alpha and beta-globin genes are coexpressed with the E. coli methionine aminopeptidase gene under the control of separate tac promoters, we have constructed a new artificial hemoglobin in which the valine residue at position 96 of the alpha chain, located in the alpha 1 beta 2 subunit interface, has been replaced by a tryptophan residue using site-directed mutagenesis. We have determined the oxygen-binding properties of this recombinant hemoglobin, r Hb (alpha 96Val-->Trp), and have used proton nuclear magnetic resonance spectroscopy to investigate its tertiary structure around the heme group and the quaternary structure in the alpha 1 beta 2 subunit interface. This artificial hemoglobin shows a low oxygen affinity, but high cooperativity in oxygen binding, and exhibits no unusual subunit dissociation when ligated. Molecular dynamics simulations suggest that the unique oxygen-binding property of r Hb (alpha 96Val-->Trp) may be due to an extra hydrogen bond between alpha 96Trp and beta 99Asp in the alpha 1 beta 2 subunit interface in the deoxy form. Despite the replacement of a small amino acid residue, valine, by a large tryptophan residue in the alpha 1 beta 2 subunit interface, this artificial hemoglobin shows very similar tertiary structure around the heme pockets and quaternary structure in the alpha 1 beta 2 subunit interface compared to those of human normal adult hemoglobin. Another unique feature of this artificial hemoglobin is that the ligated form, e.g. carbonmonoxy form, of this hemoglobin in the oxy-quaternary structure can be converted to the deoxy-like quaternary structure by the addition of an allosteric effector, inositol hexaphosphate, as well as by lowering the temperature in the absence of inositol hexaphosphate, without changing its ligation state. Thus, this recombinant hemoglobin can be used to gain new insights regarding the nature of subunit interactions in the alpha 1 beta 2 interface and the molecular basis for the allosteric mechanism of hemoglobin.
我们使用大肠杆菌表达质粒（pHE2），其中合成的人α和β珠蛋白基因在单独的tac启动子控制下与大肠杆菌甲硫氨酸氨肽酶基因共表达。我们构建了一种新的人工血红蛋白，其中位于α1β2亚基界面的α链第96位的缬氨酸残基已通过定点诱变被色氨酸残基取代。我们测定了这种重组血红蛋白r Hb（α96Val→Trp）的氧结合特性，并使用质子核磁共振光谱研究了其血红素基团周围的三级结构以及α1β2亚基界面的四级结构。这种人工血红蛋白显示出低氧亲和力，但在氧结合方面具有高协同性，并且在结合配体时没有异常的亚基解离。分子动力学模拟表明，r Hb（α96Val→Trp）独特的氧结合特性可能是由于脱氧形式下α1β2亚基界面中α96Trp和β99Asp之间存在额外的氢键。尽管在α1β2亚基界面中一个小的氨基酸残基缬氨酸被一个大的色氨酸残基取代，但与人类正常成人血红蛋白相比，这种人工血红蛋白在血红素口袋周围的三级结构以及α1β2亚基界面的四级结构非常相似。这种人工血红蛋白的另一个独特特征是，在氧四级结构中的这种血红蛋白的结合形式，例如碳氧合形式，可以通过添加变构效应剂肌醇六磷酸，以及在没有肌醇六磷酸的情况下降低温度，在不改变其结合状态的情况下转化为脱氧样四级结构。因此，这种重组血红蛋白可用于深入了解α1β2界面中亚基相互作用的本质以及血红蛋白变构机制的分子基础。
