Evolutionary and Functional Relationships in the Truncated Hemoglobin Family

Predicting function from sequence is an important goal in current biological research, and although, broad functional assignment is possible when a protein is assigned to a family, predicting functional specificity with accuracy is not straightforward. If function is provided by key structural properties and the relevant properties can be computed using the sequence as the starting point, it should in principle be possible to predict function in detail. The truncated hemoglobin family presents an interesting benchmark study due to their ubiquity, sequence diversity in the context of a conserved fold and the number of characterized members. Their functions are tightly related to O2affinity and reactivity, as determined by the association and dissociation rate constants, both of which can be predicted and analyzed using in-silico based tools. In the present work we have applied a strategy, which combines homology modeling with molecular based energy calculations, to predict and analyze function of all known truncated hemoglobins in an evolutionary context. Our results show that truncated hemoglobins present conserved family features, but that its structure is flexible enough to allow the switch from high to low affinity in a few evolutionary steps. Most proteins display moderate to high oxygen affinities and multiple ligand migration paths, which, besides some minor trends, show heterogeneous distributions throughout the phylogenetic tree, again suggesting fast functional adaptation. Our data not only deepens our comprehension of the structural basis governing ligand affinity, but they also highlight some interesting functional evolutionary trends.Fil: Bustamante, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: Radusky, Leandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Boechi, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Cálculo; ArgentinaFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: Ten Have, Arjen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Cálculo; Argentin

Gene Turnover in the Avian Globin Gene Families and Evolutionary Changes in Hemoglobin Isoform Expression

The apparent stasis in the evolution of avian chromosomes suggests that birds may have experienced relatively low rates of gene gain and loss in multigene families. To investigate this possibility and to explore the phenotypic consequences of variation in gene copy number, we examined evolutionary changes in the families of genes that encode the α- and β-type subunits of hemoglobin (Hb), the tetrameric α2β2 protein responsible for blood-O2 transport. A comparative genomic analysis of 52 bird species revealed that the size and membership composition of the α- and β-globin gene families have remained remarkably constant during approximately 100 My of avian evolution. Most interspecific variation in gene content is attributable to multiple independent inactivations of the αD-globin gene, which encodes the α-chain subunit of a functionally distinct Hb isoform (HbD) that is expressed in both embryonic and definitive erythrocytes. Due to consistent differences in O2-binding properties between HbD and the major adult-expressed Hb isoform, HbA (which incorporates products of the αA-globin gene), recurrent losses of αD-globin contribute to among-species variation in blood-O2 affinity. Analysis of HbA/HbD expression levels in the red blood cells of 122 bird species revealed high variability among lineages and strong phylogenetic signal. In comparison with the homologous gene clusters in mammals, the low retention rate for lineage-specific gene duplicates in the avian globin gene clusters suggests that the developmental regulation of Hb synthesis in birds may be more highly conserved, with orthologous genes having similar stage-specific expression profiles and similar functional properties in disparate taxa

A phylogenomic profile of globins

BACKGROUND: Globins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and «truncated» hemoglobins with a 2-over-2 instead of the canonical 3-over-3 α-helical fold. RESULTS: A census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only ~25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in ~65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematode Caenorhabditis has the most putative globins, ~33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma. CONCLUSION: Although Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals

Integration of Biological Sources: Exploring the Case of Protein Homology

Data integration is a key issue in the domain of bioin- formatics, which deals with huge amounts of heteroge- neous biological data that grows and changes rapidly. This paper serves as an introduction in the field of bioinformatics and the biological concepts it deals with, and an exploration of the integration problems a bioinformatics scientist faces. We examine ProGMap, an integrated protein homology system used by bioin- formatics scientists at Wageningen University, and several use cases related to protein homology. A key issue we identify is the huge manual effort required to unify source databases into a single resource. Un- certain databases are able to contain several possi- ble worlds, and it has been proposed that they can be used to significantly reduce initial integration efforts. We propose several directions for future work where uncertain databases can be applied to bioinformatics, with the goal of furthering the cause of bioinformatics integration

Conformational Flexibility Drives Cold Adaptation in Pseudoalteromonas haloplanktis TAC125 Globins

Significance: Temperature is one of the most important drivers in shaping protein adaptations. Many biochemical and physiological processes are influenced by temperature. Proteins and enzymes from organisms living at low temperature are less stable in comparison to high-temperature adapted proteins. The lower stability is generally due to greater conformational flexibility. Recent Advances: Adaptive changes in the structure of cold-adapted proteins may occur at subunit interfaces, distant from the active site, thus producing energy changes associated with conformational transitions transmitted to the active site by allosteric modulation, valid also for monomeric proteins in which tertiary structural changes may play an essential role. Critical Issues: Despite efforts, the current experimental and computational methods still fail to produce general principles on protein evolution, since many changes are protein and species dependent. Environmental constraints or other biological cellular signals may override the ancestral information included in the structure of the protein, thus introducing inaccuracy in estimates and predictions on the evolutionary adaptations of proteins in response to cold adaptation. Future Directions: In this review, we describe the studies and approaches used to investigate stability and flexibility in the cold-adapted globins of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. In fact, future research directions will be prescient on more detailed investigation of cold-adapted proteins and the role of fluctuations between different conformational states.Fil: Giordano, Daniela. Institute Of Biosciences And Bioresources; ItaliaFil: Boubeta, Fernando Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: di Prisco, Guido. Institute Of Biosciences And Bioresources; ItaliaFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Smulevich, Giulietta. Firenze University; ItaliaFil: Viappiani, Christiano. Università di Parma; ItaliaFil: Verde, Cinzia. Institute Of Biosciences And Bioresources; Itali

Vascular Expression of Hemoglobin Alpha in Antarctic Icefish Supports Iron Limitation as Novel Evolutionary Driver

Frigid temperatures of the Southern Ocean are known to be an evolutionary driver in Antarctic fish. For example, many fish have reduced red blood cell (RBC) concentration to minimize vascular resistance. Via the oxygen-carrying protein hemoglobin, RBCs contain the vast majority of the body’s iron, which is known to be a limiting nutrient in marine ecosystems. Since lower RBC levels also lead to reduced iron requirements, we hypothesize that low iron availability was an additional evolutionary driver of Antarctic fish speciation. Antarctic Icefish of the family Channichthyidae are known to have an extreme alteration of iron metabolism due to loss of RBCs and two iron-binding proteins, hemoglobin and myoglobin. Loss of hemoglobin is considered a maladaptive trait allowed by relaxation of predator selection since extreme adaptations are required to compensate for the loss of oxygen-carrying capacity. However, iron dependency minimization may have driven hemoglobin loss instead of a random evolutionary event. Given the variety of functions that hemoglobin serves in the endothelium, we suspected the protein corresponding to the 3’ truncated Hbα fragment (Hbα-3’f) that was not genetically excluded by icefish may still be expressed as a protein. Using whole mount confocal microscopy, we show that Hbα-3’f is expressed in the vascular endothelium of icefish retina, suggesting this Hbα fragment may still serve an important role in the endothelium. These observations support a novel hypothesis that iron minimization could have influenced icefish speciation with the loss of the iron-binding portion of Hbα in Hbα-3’f, as well as hemoglobin β and myoglobin

The Occurrence and Characterization of Hemoglobin from Different Strains of Genetically Diverse, Free-Living Frankia

Hemoglobins have been identified in root nodules of many actinorhizal plants. When cultured in vitro, the actinomycete Frankia strain CcI3 produces hemoglobin when grown with or without supplied nitrogen. The cyanobacterium, Nostoc commune, also produces hemoglobin in vitro, although only under nitrogen-fixing, microaerobic conditions, and in less than one fifth of the explored strainslspecies. The objectives of this study were to determine if Frankia strains EANlpec, ArI3, EUNlf, CcI.17, and Cc13, members of diverse genogroups, are capable of producing hemoglobin in vitro, to characterize the oxygen kinetics of the hemoglobin, and to determine the effect of nitrogen, oxygen, and carbon dioxide on the amount of hemoglobin produced. Frankia cells were disrupted under an atmosphere of carbon monoxide and the carbonmonoxy absorption spectrum of the crude extract was used to calculate the hemoglobin concentration in the cells. Hemoglobin was present in all five strains when grown on nitrogen-free (-N) or nitrogen-supplied (+N) medium. Thus it is likely that hemoglobins are produced by most Frankia strains. In four of the five strains, the hemoglobin concentrations were similar in -N and +N culture. This does not support an association between nitrogen fixation and hemoglobin expression. Hemoglobin in crude extracts from -N cultures of EANlPec was partially purified by ion exchange chromatography and then subjected to size exclusion chromatography. The molecular mass, 13.4 f 0.2 kDa (mean * SE, n = 3), is consistent with that of truncated hemoglobins, such as the Nostoc hemoglobin. The hemoglobin in other EANlPec ion exchange fractions was further purified using a CO-pressurized concentration cell with a 10 kDa exclusion membrane. The absorption spectra obtained from this sample showed carbonmonoxy and oxyhemoglobin absorption peaks typical of a hemoglobin. This same sample was also used to determine the hff value for oxygen; 131.2 f 5.8 s-\u27 (mean * SE, n = 6) and 166 + 8.2 s (mean * SE, n = 7) for the hemoglobin from -N and +N cultures, respectively. Cultures of EANlp grown at 2% and 20% 0 2 showed no effect of 0 2 on hemoglobin concentration in the +N treatments @ = 0.632). The -N treatments showed less growth than the +N treatments and the hemoglobin concentration was greater at 2% than 20% 0 2 . -Cultures of Frankia strain EANlPec and CcI3 grew more efficiently when supplied with 0.2% C02 when compared to 0.0% C02. This suggests that addition of C02 to cultures could assist in Frankia growth at low initial densities, such as isolation from root nodules. The very rapid oxygen dissociation rate of EANlp hemoglobin is two to threefold faster than that of Frankia strain CcI3 and Nostoc hemoglobin, which have been proposed to function in facilitated diffusion of oxygen. If hemoglobin is localized within the small volume of the vesicle or periphery of the cells, the concentrations in the immediate region in which it is found might approach those necessary for facilitated oxygen transport

Rice (\u3ci\u3eOryza\u3c/i\u3e) hemoglobins [version 2; peer review: 2 approved]

Hemoglobins (Hbs) corresponding to non-symbiotic (nsHb) and truncated (tHb) Hbs have been identified in rice (Oryza). This review discusses the major findings from the current studies on rice Hbs. At the molecular level, a family of the nshb genes, consisting of hb1, hb2, hb3, hb4 and hb5, and a single copy of the thb gene exist in Oryza sativa var. indica and O. sativa var. japonica, Hb transcripts coexist in rice organs and Hb polypeptides exist in rice embryonic and vegetative organs and in the cytoplasm of differentiating cells. At the structural level, the crystal structure of rice Hb1 has been elucidated, and the structures of the other rice Hbs have been modeled. Kinetic analysis indicated that rice Hb1 and 2, and possibly rice Hb3 and 4, exhibit a very high affinity for O2, whereas rice Hb5 and tHb possibly exhibit a low to moderate affinity for O2. Based on the accumulated information on the properties of rice Hbs and data from the analysis of other plant and non-plant Hbs, it is likely that Hbs play a variety of roles in rice organs, including O2-transport, O2-sensing, NO-scavenging and redox-signaling. From an evolutionary perspective, an outline for the evolution of rice Hbs is available. Rice nshb and thb genes vertically evolved through different lineages, rice nsHbs evolved into clade I and clade II lineages and rice nshbs and thbs evolved under the effect of neutral selection. This review also reveals lacunae in our ability to completely understand rice Hbs. Primary lacunae are the absence of experimental information about the precise functions of rice Hbs, the properties of modeled rice Hbs and the cis-elements and trans-acting factors that regulate the expression of rice hb genes, and the partial understanding of the evolution of rice Hbs

Plant hemoglobins: Important players at the crossroads between oxygen and nitric oxide

AbstractPlant hemoglobins constitute a diverse group of hemeproteins and evolutionarily belong to three different classes. Class 1 hemoglobins possess an extremely high affinity to oxygen and their main function consists in scavenging of nitric oxide (NO) at very low oxygen levels. Class 2 hemoglobins have a lower oxygen affinity and they facilitate oxygen supply to developing tissues. Symbiotic hemoglobins in nodules have mostly evolved from class 2 hemoglobins. Class 3 hemoglobins are truncated and represent a clade with a very low similarity to class 1 and 2 hemoglobins. They may regulate oxygen delivery at high O2 concentrations. Depending on their physical properties, hemoglobins belong either to hexacoordinate non-symbiotic or pentacoordinate symbiotic groups. Plant hemoglobins are plausible targets for improving resistance to multiple stresses

Ligand-Based Regulation of Dynamics and Reactivity of Hemoproteins

Hemoproteins include several heme-binding proteins with distinct structure and function. The presence of the heme group confers specific reactivity and spectroscopic properties to hemoproteins. In this review, we provide an overview of five families of hemoproteins in terms of dynamics and reactivity. First, we describe how ligands modulate cooperativity and reactivity in globins, such as myoglobin and hemoglobin. Second, we move on to another family of hemoproteins devoted to electron transport, such as cytochromes. Later, we consider heme-based reactivity in hemopexin, the main heme-scavenging protein. Then, we focus on heme-albumin, a chronosteric hemoprotein with peculiar spectroscopic and enzymatic properties. Eventually, we analyze the reactivity and dynamics of the most recently discovered family of hemoproteins, i.e., nitrobindins