Recombinant Gadus morhua Hemoglobin subunit alpha-2 (hba2)

What is the basic structure of Gadus morhua hemoglobin and how does the alpha-2 subunit differ from other globin chains?

Gadus morhua (Atlantic cod) hemoglobin is a tetrameric protein comprising two alpha and two beta chains. The complete hemoglobin system in Atlantic cod consists of multiple distinct globins: two alpha chains (Hb-α1 and Hb-α2) and four beta chains (Hb-β1, Hb-β2, Hb-β3, and Hb-β4) . Each alpha chain typically consists of 143 amino acids, while beta chains contain approximately 147 amino acids . The alpha-2 subunit functions as part of this quaternary structure to facilitate oxygen binding and transport.

What are the key functional domains in the hemoglobin alpha-2 subunit and how do they contribute to oxygen binding?

The hemoglobin alpha-2 subunit contains several critical functional domains:

These domains work collectively to enable the reversible binding of oxygen. Similar to other hemoglobins, the alpha-2 subunit likely participates in cooperative binding through allosteric mechanisms, where binding of oxygen to one subunit increases the affinity of remaining subunits for oxygen. This property is essential for efficient oxygen loading in gill capillaries and unloading in tissue capillaries .

How does recombinant Gadus morhua hemoglobin alpha-2 compare to naturally isolated protein?

While our search results don't provide direct comparison data between recombinant and naturally isolated Gadus morhua hemoglobin alpha-2, several general considerations apply:

Recombinant hemoglobin alpha-2 subunits are typically produced using expression systems such as E. coli or baculovirus-infected insect cells, similar to what's described for the beta-2 subunit . The recombinant protein offers several advantages over naturally isolated protein:

• Purity: Recombinant proteins typically achieve >85% purity as assessed by SDS-PAGE

• Consistency: Batch-to-batch variation is minimized compared to native protein isolation

• Scalability: Larger quantities can be produced for extensive research applications

• Modification potential: Specific tags can be incorporated for downstream applications

What polymorphisms exist in Gadus morhua hemoglobin alpha-2 and how do they impact protein function?

What we can infer is that any polymorphisms in the alpha-2 subunit could potentially affect:

• Oxygen binding affinity: Changes in key residues may alter the affinity for oxygen

• Subunit interactions: Polymorphisms at subunit interfaces could affect cooperativity

• Temperature sensitivity: Similar to the beta chain polymorphisms, alpha chain variations may influence temperature-dependent oxygen binding properties

• pH sensitivity: Alterations in specific residues could affect the Bohr effect (pH-dependent oxygen affinity)

The polymorphisms in the beta-1 subunit (Met55Val and Lys62Ala) demonstrate how single amino acid changes can significantly impact functional properties. These replacements affect the quaternary structure and electrostatic features of the hemoglobin molecule, thereby altering oxygen-binding properties . Similar mechanisms likely apply to any polymorphisms present in the alpha-2 subunit.

How do the hemoglobin variants in Gadus morhua relate to environmental adaptation?

The hemoglobin system in Atlantic cod shows remarkable adaptation to environmental conditions. While specific data on alpha-2 adaptations is limited in the search results, the beta chain polymorphisms provide insight into how hemoglobin variants facilitate environmental adaptation:

Cod populations inhabiting different environments show distinct distributions of hemoglobin variants. Fish in cold Arctic waters and the low-oxygen Baltic Sea predominantly possess the high oxygen affinity Val55-Ala62 haplotype in their beta-1 subunit, while the temperature-insensitive Met55-Lys62 haplotype is more common in southern populations .

This distribution correlates with physiological preferences:

• HbI-2/2 cod (with Val55-Ala62) prefer lower temperatures (8.2°C)

• HbI-1/1 cod (with Met55-Lys62) prefer higher temperatures (15.4°C)

The functional basis for these adaptations lies in the oxygen-binding properties of the variants:

• At low temperatures (<12°C), the HbI-2/2 phenotype shows higher oxygen-binding affinity

• At higher temperatures (up to 20°C), the HbI-1/1 phenotype exhibits greater oxygen affinity

These adaptations illustrate how hemoglobin polymorphisms contribute to the species' ability to inhabit waters with varying temperature and oxygen regimes. Similar adaptive mechanisms likely exist for any polymorphisms in the alpha-2 subunit, potentially working in concert with beta chain variants to fine-tune oxygen transport capabilities.

What molecular mechanisms underlie the functional differences between hemoglobin variants?

The molecular mechanisms responsible for functional differences between hemoglobin variants involve alterations in:

• Quaternary structure: The Met55Val and Lys62Ala replacements in the beta-1 chain are located at crucial positions that affect subunit interfaces and the haem pocket. Three-dimensional modeling revealed that these replacements modify the quaternary structure of the hemoglobin molecule .

• Electrostatic properties: The Lys62Ala substitution changes the electrostatic features of the haem pocket, affecting oxygen binding .

• Water molecule interactions: GRID analysis of the polymorphic variants demonstrated differences in the interaction of water molecules near the distal histidine of the beta haem pocket, which influences ligand binding properties .

• Heme pocket environment: The polymorphisms alter the hydrophobic and polar characteristics of the distal environment of the haem pocket, as investigated through GRID program analysis .

Interestingly, similar molecular mechanisms facilitating oxygen binding have been found in avian species adapted to high altitudes, illustrating convergent evolution between water-breathing and air-breathing vertebrates in response to reduced environmental oxygen availability .

What are the optimal protocols for reconstitution and handling of recombinant Gadus morhua hemoglobin subunits?

Based on the available information for recombinant Gadus morhua hemoglobin subunits, the following protocols are recommended:

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended as default)

• Aliquot for long-term storage

Storage Recommendations:

• Store at -20°C, or at -80°C for extended storage

• Avoid repeated freezing and thawing

• Working aliquots can be stored at 4°C for up to one week

Shelf Life:

• Liquid form: approximately 6 months at -20°C/-80°C

• Lyophilized form: approximately 12 months at -20°C/-80°C

These recommendations apply to both E. coli-expressed and baculovirus-expressed recombinant proteins, though expression system-specific variations may exist.

What experimental techniques are most effective for studying Gadus morhua hemoglobin subunit interactions and oxygen binding properties?

Several techniques can effectively characterize hemoglobin subunit interactions and oxygen binding properties:

For Subunit Interactions:

• Size-exclusion chromatography: To examine tetramer formation and stability

• Isothermal titration calorimetry (ITC): For quantitative analysis of binding energetics between subunits

• Cross-linking studies: To investigate proximity relationships between subunits

• Homology modeling: As demonstrated in the research, comparative modeling using templates from related fish species (e.g., Trematomus bernacchii, Trematomus newnesi, Thunnus thynnus, and Oncorhynchus mykiss) can provide valuable structural insights

For Oxygen Binding Properties:

• Oxygen equilibrium curves: To determine P50 values and cooperativity coefficients

• Temperature-dependent binding studies: Particularly important given the temperature-sensitive properties of cod hemoglobin variants

• pH-dependent binding studies: To characterize the Bohr effect

• GRID analysis: To investigate the hydrophobic and polar characteristics of the haem pocket environment, as demonstrated in previous research

Verification Methods:

• SDS-PAGE: For purity assessment (>85% purity is typically achieved for recombinant proteins)

• Spectroscopic techniques: To analyze structural integrity and ligand binding

• Mass spectrometry: For precise molecular weight determination and identification of post-translational modifications

How can researchers effectively express and purify recombinant Gadus morhua hemoglobin alpha-2?

Based on the available information for recombinant hemoglobin production, researchers can use several expression systems:

Expression Systems:

• E. coli: A commonly used system for recombinant Gadus morhua hemoglobin subunits, as seen with the beta-2 subunit

• Baculovirus-infected insect cells: Another effective system for producing functional hemoglobin subunits

• Yeast expression: Potentially useful for proteins requiring eukaryotic post-translational modifications

• Wheat germ: Used for human hemoglobin subunit alpha expression, suggesting potential applicability for fish hemoglobins

Purification Strategy:

• Affinity chromatography: Using tags that can be incorporated during the expression process

• Ion-exchange chromatography: Based on the charge properties of the protein

• Size-exclusion chromatography: For final polishing and buffer exchange

Quality Control:

• SDS-PAGE: To assess purity (target >85%)

• Western blotting: For specific identification

• Mass spectrometry: For accurate mass determination

• Functional assays: To confirm oxygen binding capability

Tag Options:
The tag type is typically determined during the manufacturing process. Options may include:

• His-tag for metal affinity purification

• GST-tag for glutathione affinity purification

• Avi-tag for biotinylation, as mentioned for other recombinant proteins

How does the structure of Gadus morhua hemoglobin alpha-2 compare to hemoglobins from other species?

While detailed structural information specific to Gadus morhua hemoglobin alpha-2 is limited in the search results, we can infer comparative aspects based on the structural modeling approaches described:

The structure of fish hemoglobins, including those from Atlantic cod, shares the basic globin fold found across species but exhibits specific adaptations. Structural modeling of cod hemoglobin used templates from several fish species that shared the highest sequence identity:

• Antarctic fish: Trematomus bernacchii (PDB code 1HBH)

• Dusky notothen: Trematomus newnesi (PDB code 2AA1)

• Bluefin tuna: Thunnus thynnus (PDB code 1V4W)

• Rainbow trout: Oncorhynchus mykiss (PDB code 1OUT)

Each chain model was built using comparative modeling with the Modeller program, and the quality was assessed based on the Modeller objective function and Procheck stereochemical criteria .

The human hemoglobin alpha subunit consists of 142 amino acids , while fish alpha chains are typically around 143 amino acids . This similarity suggests conservation of the basic globin fold structure across vertebrates, despite sequence variations that confer species-specific functional properties.

The three-dimensional modeling of cod hemoglobin revealed that polymorphic residues in the beta chain are located at crucial positions affecting the alpha-beta subunit interface and haem pocket . Similar strategic positioning of functionally important residues likely exists in the alpha-2 subunit.

What functional differences exist between hemoglobin variants in different fish species adapted to various environmental conditions?

Hemoglobin variants across fish species show adaptations to diverse environmental conditions:

• Temperature adaptations:

  • Atlantic cod hemoglobin variants show differential temperature preferences, with the HbI-2/2 phenotype preferring lower temperatures (8.2°C) and the HbI-1/1 phenotype preferring higher temperatures (15.4°C)

  • Antarctic fish species (e.g., Trematomus bernacchii) have hemoglobins adapted to consistently cold environments

• Oxygen availability adaptations:

  • Cod populations in the low-oxygen Baltic Sea have high oxygen affinity variants (Val55-Ala62 haplotype)

  • Species inhabiting oxygen-variable environments often possess multiple hemoglobin isoforms with different oxygen affinities

• Convergent evolution:

  • The molecular mechanism facilitating oxygen binding in cod is remarkably similar to that found in avian species adapted to high altitudes, illustrating convergent evolution between water-breathing and air-breathing vertebrates

These adaptations involve similar molecular mechanisms across species:

• Modifications of subunit interfaces affecting cooperativity

• Alterations of the electrostatic features of the haem pocket

• Changes in the interaction networks stabilizing different conformational states

The distinct distributions of functionally different hemoglobin variants across populations and species highlight the importance of these proteins in environmental adaptation.

How do the functional regions of hemoglobin alpha-2 contribute to species-specific adaptations?

While specific information about functional regions in Gadus morhua hemoglobin alpha-2 is limited in the search results, we can infer their roles in adaptation based on the documented polymorphisms in the beta chains and general hemoglobin structure-function relationships:

• Subunit interface regions: Amino acid replacements at subunit interfaces can affect the stability of different quaternary structures (R and T states), influencing oxygen binding cooperativity. In cod beta-1 subunit, the Met55Val replacement occurs at the α1β1 subunit interface .

• Heme pocket residues: Modifications in the heme pocket environment directly influence oxygen binding affinity. The Lys62Ala replacement in cod beta-1 affects the haem pocket electrostatic features .

• Allosteric effector binding sites: Regions that interact with allosteric effectors (e.g., protons, chloride ions) determine the response to environmental factors. These regions likely contribute to the temperature sensitivity of oxygen binding in different cod hemoglobin variants.

• Surface residues: Surface-exposed residues can affect solubility, stability, and interactions with other cellular components, potentially contributing to adaptation to specific cellular environments.

These functional regions likely work in concert to produce the unique adaptive properties observed in different fish species and populations. The strategic positioning of polymorphic residues at functionally critical locations, as seen in the beta-1 chain, suggests a similar pattern may exist in the alpha-2 subunit.

How can recombinant Gadus morhua hemoglobin subunits be used in studies of climate change impacts on marine species?

Recombinant Gadus morhua hemoglobin subunits offer valuable tools for studying climate change impacts on marine species:

• Temperature adaptation studies:

  • The temperature-dependent properties of cod hemoglobin variants make them excellent models for investigating physiological responses to warming oceans

  • Researchers can use recombinant subunits to reconstruct hemoglobin tetramers with different combinations of variants to assess temperature effects on oxygen transport efficiency

• Predictive modeling:

  • The distinct distribution of hemoglobin variants across temperature gradients suggests that global warming could significantly affect the biogeography of Atlantic cod

  • Recombinant proteins allow researchers to experimentally test predictions about how changing temperature and oxygen regimes will affect different cod populations

• Experimental evolution studies:

  • By introducing specific mutations into recombinant hemoglobin subunits, researchers can investigate potential evolutionary responses to changing marine environments

• Physiological tolerance limits:

  • Researchers can use recombinant hemoglobins to determine the physiological limits of different variants under projected climate change scenarios

  • This information can inform conservation strategies for vulnerable populations

Such applications require careful experimental design and should integrate multiple approaches, including in vitro functional studies with recombinant proteins, in vivo physiological assessments, and ecological modeling.

What methodologies can be employed to study the effect of post-translational modifications on hemoglobin function?

Post-translational modifications (PTMs) can significantly affect hemoglobin function. Several methodologies can be employed to study these effects:

• Mass spectrometry:

  • Liquid chromatography-mass spectrometry (LC-MS/MS) for comprehensive PTM mapping

  • MALDI-TOF MS for more targeted analysis

  • Hydrogen-deuterium exchange MS (HDX-MS) to probe structural changes induced by PTMs

• Site-directed mutagenesis:

  • Generation of recombinant proteins with mutations at potential PTM sites

  • Comparison of wild-type and mutant proteins to assess functional impact

• Expression system selection:

  • E. coli for producing unmodified proteins

  • Eukaryotic systems (baculovirus , yeast , wheat germ ) for proteins requiring specific PTMs

• Functional assays:

  • Oxygen equilibrium curves to assess effects on oxygen binding affinity and cooperativity

  • Stability studies to determine if PTMs affect protein resilience under different conditions

• Structural analysis:

  • X-ray crystallography to determine 3D structural changes

  • Modeling approaches similar to those used for studying hemoglobin polymorphisms

A comprehensive approach would involve producing recombinant proteins with and without specific PTMs, characterizing them structurally and functionally, and correlating findings with physiological performance under various environmental conditions.

How can molecular dynamics simulations enhance our understanding of hemoglobin structure-function relationships?

Molecular dynamics (MD) simulations provide powerful tools for investigating hemoglobin structure-function relationships at the atomic level:

• Conformational dynamics:

  • MD simulations can reveal transitions between R (relaxed, high affinity) and T (tense, low affinity) states

  • Time-dependent fluctuations in key regions can be correlated with functional properties

• Allosteric mechanisms:

  • Simulations can elucidate pathways of communication between subunits

  • Energy propagation through the protein can be traced following oxygen binding/release events

• Effect of polymorphisms:

  • Virtual mutagenesis in MD simulations can predict functional impacts of specific amino acid replacements

  • Comparing simulations of different natural variants can explain observed functional differences

• Environmental factor effects:

  • Temperature, pH, and salt concentration effects can be systematically investigated

  • Results can explain the adaptive significance of different variants in various environmental conditions

• Water interactions:

  • Similar to the GRID analysis used for studying the haem pocket environment , MD simulations can provide dynamic information about water molecule interactions and their functional significance

The methodological approach would involve:

• Building accurate homology models based on available structures

• Setting up simulations with appropriate force fields and solvent models

• Running multiple replicate simulations to ensure statistical significance

• Analyzing trajectories for key interactions, conformational changes, and energetic parameters

What quality control measures should be implemented when working with recombinant Gadus morhua hemoglobin subunits?

Comprehensive quality control measures for recombinant hemoglobin subunits include:

• Purity assessment:

  • SDS-PAGE analysis should confirm >85% purity

  • Western blot verification of identity

  • Mass spectrometry confirmation of molecular weight

• Structural integrity:

  • Circular dichroism (CD) spectroscopy to confirm proper secondary structure

  • Fluorescence spectroscopy to assess tertiary structure

  • UV-visible spectroscopy to examine heme environment

• Functional verification:

  • Oxygen binding assays to confirm functional activity

  • Cooperativity measurements

  • Temperature-dependent functional studies

• Stability testing:

  • Thermal stability assessments

  • Storage stability monitoring at different temperatures

  • Freeze-thaw stability tests

• Lot-to-lot consistency:

  • Comparison of key parameters between production batches

  • Standardized reference samples for comparative analysis

Each recombinant protein should be accompanied by a Certificate of Analysis documenting key quality parameters and meeting pre-established acceptance criteria.

What are common challenges in experimental design when studying hemoglobin polymorphisms, and how can they be addressed?

Research on hemoglobin polymorphisms presents several experimental challenges:

• Challenge: Isolating individual effects of specific polymorphisms when multiple variations exist
  Solution:

  • Use recombinant proteins with single mutations for systematic analysis

  • Apply statistical methods like multivariate analysis to dissect the contribution of each polymorphism

  • Develop experimental designs that control for confounding variables

• Challenge: Replicating physiologically relevant conditions in vitro
  Solution:

  • Include appropriate allosteric effectors (pH buffers, chloride, organophosphates)

  • Test across physiologically relevant temperature ranges

  • Consider using whole blood or red cell lysates alongside purified proteins

• Challenge: Correlating molecular properties with ecological significance
  Solution:

  • Integrate laboratory studies with field observations

  • Use biogeographical data on variant distribution

  • Develop predictive models linking molecular properties to environmental adaptation

• Challenge: Tetrameric hemoglobin assembly from recombinant subunits
  Solution:

  • Optimize reconstitution protocols for proper tetramer formation

  • Verify assembly using size-exclusion chromatography

  • Ensure correct heme incorporation and oxidation state

• Challenge: Distinguishing genetic adaptation from phenotypic plasticity
  Solution:

  • Compare populations from different environments but with similar genetic backgrounds

  • Conduct common garden experiments

  • Integrate genomic, proteomic, and physiological approaches

What are the most reliable assays for determining functional properties of recombinant hemoglobin variants?

Several reliable assays can determine the functional properties of recombinant hemoglobin variants:

• Oxygen equilibrium curves:

  • Tonometry methods to determine P50 (oxygen pressure at 50% saturation)

  • Hill coefficient calculation for cooperativity assessment

  • Temperature-controlled measurements to determine thermal sensitivity

• Kinetic measurements:

  • Stopped-flow techniques for oxygen association/dissociation rates

  • Flash photolysis for ligand binding kinetics

  • Temperature-jump experiments for conformational change rates

• Structural stability assays:

  • Differential scanning calorimetry (DSC) for thermal stability

  • Chemical denaturation monitored by spectroscopic techniques

  • Limited proteolysis for domain stability assessment

• Allosteric effector response:

  • pH titration curves to characterize the Bohr effect

  • Binding studies with organic phosphates (e.g., ATP, GTP)

  • Anion (chloride) effect measurements

• Comparative functional genomics:

  • Correlation of sequence variations with functional properties

  • Structure-based predictions validated by experimental measurements

  • Evolutionary analysis of sequence-function relationships

For most comprehensive understanding, multiple complementary techniques should be employed, and results should be interpreted in the context of the physiological environment of the species.