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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL087216-06A1
Application #
8760953
Study Section
Genetic Variation and Evolution Study Section (GVE)
Program Officer
Luksenburg, Harvey
Project Start
2006-07-01
Project End
2018-04-30
Budget Start
2014-08-01
Budget End
2015-04-30
Support Year
6
Fiscal Year
2014
Total Cost
$365,414
Indirect Cost
$110,793
Name
University of Nebraska Lincoln
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
555456995
City
Lincoln
State
NE
Country
United States
Zip Code
68583
Storz, Jay F (2016) Gene Duplication and Evolutionary Innovations in Hemoglobin-Oxygen Transport. Physiology (Bethesda) 31:223-32
Storz, Jay F (2016) Causes of molecular convergence and parallelism in protein evolution. Nat Rev Genet 17:239-50
Jensen, Birgitte; Storz, Jay F; Fago, Angela (2016) Bohr effect and temperature sensitivity of hemoglobins from highland and lowland deer mice. Comp Biochem Physiol A Mol Integr Physiol 195:10-4
Storz, Jay F (2016) Hemoglobin-oxygen affinity in high-altitude vertebrates: is there evidence for an adaptive trend? J Exp Biol 219:3190-3203
Galen, Spencer C; Natarajan, Chandrasekhar; Moriyama, Hideaki et al. (2015) Contribution of a mutational hot spot to hemoglobin adaptation in high-altitude Andean house wrens. Proc Natl Acad Sci U S A 112:13958-63
Storz, Jay F; Natarajan, Chandrasekhar; Moriyama, Hideaki et al. (2015) Oxygenation properties and isoform diversity of snake hemoglobins. Am J Physiol Regul Integr Comp Physiol 309:R1178-91
Natarajan, Chandrasekhar; Hoffmann, Federico G; Lanier, Hayley C et al. (2015) Intraspecific polymorphism, interspecific divergence, and the origins of function-altering mutations in deer mouse hemoglobin. Mol Biol Evol 32:978-97
Storz, Jay F; Bridgham, Jamie T; Kelly, Scott A et al. (2015) Genetic approaches in comparative and evolutionary physiology. Am J Physiol Regul Integr Comp Physiol 309:R197-214
Lui, Mikaela A; Mahalingam, Sajeni; Patel, Paras et al. (2015) High-altitude ancestry and hypoxia acclimation have distinct effects on exercise capacity and muscle phenotype in deer mice. Am J Physiol Regul Integr Comp Physiol 308:R779-91
Scott, Graham R; Elogio, Todd S; Lui, Mikaela A et al. (2015) Adaptive Modifications of Muscle Phenotype in High-Altitude Deer Mice Are Associated with Evolved Changes in Gene Regulation. Mol Biol Evol 32:1962-76

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