Epistasis Among Adaptive Mutations in Deer Mouse Hemoglobin

Epistatic interactions between mutant sites in the same protein can exert a strong influence on pathways of molecular evolution. We performed protein engineering experiments that revealed pervasive epistasis among segregating amino acid variants that contribute to adaptive functional variation in deer mouse hemoglobin (Hb). Amino acid mutations increased or decreased Hb-O2 affinity depending on the allelic state of other sites. Structural analysis revealed that epistasis for Hb-O2 affinity and allosteric regulatory control is attributable to indirect interactions between structurally remote sites. The prevalence of sign epistasis for fitness-related biochemical phenotypes has important implications for the evolutionary dynamics of protein polymorphism in natural populations

Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix. Corrigendum

In the article by Inoguchi et al. (2013) the affiliation for two of the authors, Angela Fago and Roy E. Weber, was given incorrectly. The correct affiliation is Zoophysiology, Department of Bioscience, Aarhus University, Aarhus, Denmark

Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix

The deer mouse, Peromyscus maniculatus, exhibits altitude-associated variation in hemoglobin oxygen affinity. To examine the structural basis of this functional variation, the structure of the hemoglobin was solved. Recombinant hemoglobin was expressed in Escherichia coli and was purified by ion-exchange chromatography. Recombinant hemoglobin was crystallized by the hangingdrop vapor-diffusion method using polyethylene glycol as a precipitant. The obtained orthorhombic crystal contained two subunits in the asymmetric unit. The refined structure was interpreted as the aquo-met form. Structural comparisons were performed among hemoglobins from deer mouse, house mouse and human. In contrast to human hemoglobin, deer mouse hemoglobin lacks the hydrogen bond between α1Trp14 in the A helix and α1Thr67 in the E helix owing to the Thr67Ala substitution. In addition, deer mouse hemoglobin has a unique hydrogen bond at the α1β1 interface between residues α1Cys34 and β1Ser128

Structural insights into the mechanism defining substrate affinity in Arabidopsis thaliana dUTPase: the role of tryptophan 93 in ligand orientation

Background: Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) hydrolyzes dUTP to dUMP and pyrophosphate to maintain the cellular thymine-uracil ratio. dUTPase is also a target for cancer chemotherapy. However, the mechanism defining its substrate affinity remains unclear. Sequence comparisons of various dUTPases revealed that Arabidopsis thaliana dUTPase has a unique tryptophan at position 93, which potentially contributes to its degree of substrate affinity. To better understand the roles of tryptophan 93, A. thaliana dUTPase was studied. Results: Enzyme assays showed that A. thaliana dUTPase belongs to a high-affinity group of isozymes, which also includes the enzymes from Escherichia coli and Mycobacterium tuberculosis. Enzymes from Homo sapiens and Saccharomyces cerevisiae are grouped as low-affinity dUTPases. The structure of the homo-trimeric A. thaliana dUTPase showed three active sites, each with a different set of ligand interactions between the amino acids and water molecules. On an α-helix, tryptophan 93 appears to keep serine 89 in place via a water molecule and to specifically direct the ligand. Upon being oriented in the active site, the C-terminal residues close the active site to promote the reaction. Conclusions: In the high-affinity group, the prefixed direction of the serine residues was oriented by a positively charged residue located four amino acids away, while low-affinity enzymes possess small hydrophobic residues at the corresponding sites

Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix

The deer mouse, Peromyscus maniculatus, exhibits altitude-associated variation in hemoglobin oxygen affinity. To examine the structural basis of this functional variation, the structure of the hemoglobin was solved. Recombinant hemoglobin was expressed in Escherichia coli and was purified by ion-exchange chromatography. Recombinant hemoglobin was crystallized by the hangingdrop vapor-diffusion method using polyethylene glycol as a precipitant. The obtained orthorhombic crystal contained two subunits in the asymmetric unit. The refined structure was interpreted as the aquo-met form. Structural comparisons were performed among hemoglobins from deer mouse, house mouse and human. In contrast to human hemoglobin, deer mouse hemoglobin lacks the hydrogen bond between α1Trp14 in the A helix and α1Thr67 in the E helix owing to the Thr67Ala substitution. In addition, deer mouse hemoglobin has a unique hydrogen bond at the α1β1 interface between residues α1Cys34 and β1Ser128

Studies of functional regulations in allosteric proteins

Structures and functions of proteins are closely related, and variations in amino acid sequences are a result of their evolutionary adaptations. Since substitutions are not only found at the active site but also on the distal surfaces of the proteins; and a majority of biological reactions occur in an aqueous environment, the contribution of water molecules to protein function will be discussed. Comparison of proteins from different populations or species is advantageous to our understanding of the molecular mechanism of adaptation. For this purpose, I compared mutations observed in natural variants with functional properties of protein. In this study, hemoglobin and deoxyuridine triphosphatase (dUTPase) were chosen as model systems. Hemoglobin is an oxygen-transporting protein that that maintains a low redox-level. Hemoglobins from deer mice living at highland and lowland populations were compared. The surface interactions between amino acids and water molecules were observed to govern the allostery. dUTPase is a hydrolase that maintains a cellular balanced in pyrimidine. Comparative studies of dUTPases from various species, including Arabidopsis, revealed a contribution of water molecules in the enzyme’s substrate affinity. These studies show that alteration of hydrogen bonding is a key factor contributing to functional variations

Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix. Corrigendum

In the article by Inoguchi et al. (2013) the affiliation for two of the authors, Angela Fago and Roy E. Weber, was given incorrectly. The correct affiliation is Zoophysiology, Department of Bioscience, Aarhus University, Aarhus, Denmark

Genome-wide comparison inferred the origin and evolution of B-cell epitopes on the proteins of human influenza A virus

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