A fundamental question in evolutionary genetics concerns the roles of mutational pleiotropy and epistasis in shaping trajectories of protein evolution. A powerful means of addressing this question involves the use of site-directed mutagenesis to explore the mutational landscape of protein function in experimentally defined regions of sequence space. Here we describe a plan to evaluate how pleiotropic trade-offs and epistatic interactions influence the selective accessibility of alternative mutational pathways during the adaptive functional evolution of mammalian hemoglobin (Hb). Using ancestral protein resurrection in conjunction with a combinatorial protein-engineering approach based on site-directed mutagenesis, we will examine the structural and functional effects of sequential mutational steps in all possible pathways that lead to the evolution of an increased Hb-O2 affinity in mammals that have adapted to environmental hypoxia. To evaluate the influence of pleiotropy and epistasis on adaptive protein evolution, we will examine the molecular basis of adaptive changes in Hb function at several different levels of divergence between pairs of mammalian taxa that have evolved different Hb-O2 affinities. The experimental approach integrates biochemical and biophysical examinations of the effects of specific mutations in recombinantly expressed Hbs. One of the primary innovations of this project is that we have developed an expression vector system that allows us to synthesize recombinant Hb in E. coli host cells. The research is designed to accomplish the following aims: (1) Identify the specific mutations that contribute to evolutionary changes in Hb function, and determine the relative contributions of additive and epistatic effects, and (2) Identify and characterize the biochemical/biophysical mechanisms responsible for observed pleiotropic effects and epistatic interactions. Accomplishing these two aims will reveal the specific mutations that have contributed to adaptive modifications of protein function, and will elucidate the specific biochemical/biophysical mechanisms by which pleiotropy and epistasis affect the selective accessibility of evolutionary pathways. By using Hb as a model molecule, we can leverage extremely rich sources of information about structure-function relationships to gain insights into mechanism. By directly measuring the structural and functional effects of causative mutations, our experimental results will provide answers to fundamental questions about molecular adaptation and mechanisms of protein evolution.

Public Health Relevance

Public Health Relevance Statement This research project is designed to reveal the specific mutations that are responsible for functional changes in hemoglobin (Hb), a critically important protein that is responsible for circulatory oxygen-transport. Our insights into the functional effects of Hb mutations (both singly and in combination) may shed light on the molecular mechanisms underlying certain Hb pathologies, and may also help guide the design of recombinant Hbs for use as cell-free O2- carriers in transfusion medicine. Indeed, accounting for nonadditive effects of affinity-altering mutations is central to strategies for designing Hb-based O2-carriers.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Genetic Variation and Evolution Study Section (GVE)
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Luksenburg, Harvey
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University of Nebraska Lincoln
Schools of Arts and Sciences
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Gaudry, Michael J; Storz, Jay F; Butts, Gary Tyler et al. (2014) Repeated evolution of chimeric fusion genes in the ?-globin gene family of laurasiatherian mammals. Genome Biol Evol 6:1219-34
Cheviron, Zachary A; Connaty, Alex D; McClelland, Grant B et al. (2014) Functional genomics of adaptation to hypoxic cold-stress in high-altitude deer mice: transcriptomic plasticity and thermogenic performance. Evolution 68:48-62
Cheviron, Zachary A; Natarajan, Chandrasekhar; Projecto-Garcia, Joana et al. (2014) Integrating evolutionary and functional tests of adaptive hypotheses: a case study of altitudinal differentiation in hemoglobin function in an Andean Sparrow, Zonotrichia capensis. Mol Biol Evol 31:2948-62
Tufts, Danielle M; Revsbech, Inge G; Cheviron, Zachary A et al. (2013) Phenotypic plasticity in blood-oxygen transport in highland and lowland deer mice. J Exp Biol 216:1167-73
Natarajan, Chandrasekhar; Inoguchi, Noriko; Weber, Roy E et al. (2013) Epistasis among adaptive mutations in deer mouse hemoglobin. Science 340:1324-7
Damsgaard, Christian; Storz, Jay F; Hoffmann, Federico G et al. (2013) Hemoglobin isoform differentiation and allosteric regulation of oxygen binding in the turtle, Trachemys scripta. Am J Physiol Regul Integr Comp Physiol 305:R961-7
Weber, Roy E; Fago, Angela; Malte, Hans et al. (2013) Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin. Am J Physiol Regul Integr Comp Physiol 305:R300-12
Storz, Jay F; Opazo, Juan C; Hoffmann, Federico G (2013) Gene duplication, genome duplication, and the functional diversification of vertebrate globins. Mol Phylogenet Evol 66:469-78
Revsbech, Inge G; Tufts, Danielle M; Projecto-Garcia, Joana et al. (2013) Hemoglobin function and allosteric regulation in semi-fossorial rodents (family Sciuridae) with different altitudinal ranges. J Exp Biol 216:4264-71
Inoguchi, Noriko; Oshlo, Jake R; Natarajan, Chandrasekhar et al. (2013) Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:393-8

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