Maillett David H, Simplaceanu Virgil, Shen Tong-Jian, Ho Nancy T, Olson John S, Ho Chien
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
Biochemistry. 2008 Oct 7;47(40):10551-63. doi: 10.1021/bi800816v. Epub 2008 Sep 13.
Protein engineering strategies seek to develop a hemoglobin-based oxygen carrier with optimized functional properties, including (i) an appropriate O 2 affinity, (ii) high cooperativity, (iii) limited NO reactivity, and (iv) a diminished rate of auto-oxidation. The mutations alphaL29F, alphaL29W, alphaV96W and betaN108K individually impart some of these traits and in combinations produce hemoglobin molecules with interesting ligand-binding and allosteric properties. Studies of the ligand-binding properties and solution structures of single and multiple mutants have been performed. The aromatic side chains placed in the distal-heme pocket environment affect the intrinsic ligand-binding properties of the mutated subunit itself, beyond what can be explained by allostery, and these changes are accompanied by local structural perturbations. In contrast, hemoglobins with mutations in the alpha 1beta 1 and alpha 1beta 2 interfaces display functional properties of both "R"- and "T"-state tetramers because the equilibrium between quaternary states is altered. These mutations are accompanied by global structural perturbations, suggesting an indirect, allostery-driven cause for their effects. Combinations of the distal-heme pocket and interfacial mutations exhibit additive effects in both structural and functional properties, contribute to our understanding of allostery, and advance protein-engineering methods for manipulating the O 2 binding properties of the hemoglobin molecule.
蛋白质工程策略旨在开发一种具有优化功能特性的基于血红蛋白的氧载体，这些特性包括：（i）适当的氧气亲和力；（ii）高协同性；（iii）有限的一氧化氮反应性；以及（iv）降低的自动氧化速率。αL29F、αL29W、αV96W和βN108K这几个突变单独赋予了其中一些特性，并且这些突变组合会产生具有有趣的配体结合和别构特性的血红蛋白分子。已经对单个和多个突变体的配体结合特性和溶液结构进行了研究。置于远侧血红素口袋环境中的芳香族侧链会影响突变亚基自身的内在配体结合特性，这种影响超出了别构效应所能解释的范围，并且这些变化伴随着局部结构扰动。相比之下，在α1β1和α1β2界面处有突变的血红蛋白表现出“R”态和“T”态四聚体的功能特性，因为四级状态之间的平衡发生了改变。这些突变伴随着整体结构扰动，表明其效应是由间接的、别构驱动的原因导致的。远侧血红素口袋突变和界面突变的组合在结构和功能特性上表现出累加效应，有助于我们理解别构现象，并推动了用于操纵血红蛋白分子氧结合特性的蛋白质工程方法的发展。
