title: Interfacial and distal-heme pocket mutations exhibit additive effects on the structure and function of hemoglobin.
abstract: 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.
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: David H
authors.initials: DH
authors.lastname: Maillett
authors.forename: Virgil
authors.initials: V
authors.lastname: Simplaceanu
authors.forename: Tong-Jian
authors.initials: TJ
authors.lastname: Shen
authors.forename: Nancy T
authors.initials: NT
authors.lastname: Ho
authors.forename: John S
authors.initials: JS
authors.lastname: Olson
authors.forename: Chien
authors.initials: C
authors.lastname: Ho
mesh_terms: D006418:Heme / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D006454:Hemoglobins; D011994:Recombinant Proteins; D006418:Heme; D010100:Oxygen
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