Recombinant Dasyatis akajei Hemoglobin subunit beta (HBB)

What is the structural organization of Dasyatis akajei hemoglobin and how does it compare to human hemoglobin?

Key structural differences include:

Despite these structural differences, the functional properties, including oxygen affinity, haem-haem interaction (cooperativity), Bohr effect, and organic phosphate effect are only moderately reduced compared to human HbA .

What expression systems are suitable for producing recombinant Dasyatis akajei HBB?

While the search results don't specifically address expression systems for D. akajei HBB, research on recombinant hemoglobins provides applicable methodologies. Escherichia coli represents the most commonly used expression system for recombinant hemoglobins, offering high yield and relatively straightforward genetic manipulation .

For optimal expression of recombinant D. akajei HBB, researchers should consider:

• Codon optimization for the expression host (particularly important for heterologous expression of fish proteins in bacterial systems)

• N-terminal modifications similar to those used for human hemoglobin expression, such as the V1M mutation to facilitate methionine aminopeptidase processing

• Co-expression with hemoglobin alpha subunit to promote proper folding and assembly

• Inclusion of heme synthesis promoters or supplementation with exogenous hemin

Expression in E. coli typically involves:

• Cloning the optimized D. akajei HBB gene into an expression vector with an inducible promoter

• Transformation into an E. coli strain optimized for protein expression

• Induction of protein expression under controlled conditions

• Cell lysis and initial purification steps

Alternative expression systems including yeast, insect cells, or mammalian cells might offer advantages for complex post-translational modifications or when bacterial expression results in inclusion bodies.

What purification strategies are effective for recombinant Dasyatis akajei HBB?

Based on established methods for recombinant hemoglobins, a multi-step purification process would be recommended:

• Initial clarification: Centrifugation of cell lysate followed by filtration to remove cell debris

• Capture step: Anion exchange chromatography (e.g., Q-Sepharose) at pH 8.0-8.5

• Intermediate purification: Hydrophobic interaction chromatography

• Polishing step: Size exclusion chromatography to separate tetrameric hemoglobin from dimers, monomers, or aggregates

For recombinant hemoglobins expressed in E. coli, additional considerations include:

• Incorporation of a polyhistidine tag for initial purification by immobilized metal affinity chromatography (IMAC)

• Potential need for refolding protocols if the protein forms inclusion bodies

• Reconstitution with heme if the protein is expressed in the apo form

Throughout purification, spectroscopic analysis (UV-visible spectroscopy at 280 nm, 415 nm, 540 nm, and 575 nm) should be used to track hemoglobin content and assess heme incorporation.

What analytical methods can characterize recombinant Dasyatis akajei HBB properties?

Multiple complementary techniques are necessary to thoroughly characterize recombinant D. akajei HBB:

• Structural characterization:

  • SDS-PAGE and native PAGE for purity and oligomeric state

  • Mass spectrometry for precise molecular weight and post-translational modifications

  • Circular dichroism for secondary structure content

  • X-ray crystallography for high-resolution structural determination, similar to the approach used for native D. akajei hemoglobin

• Functional characterization:

  • UV-Vis spectroscopy to confirm heme incorporation and oxidation state

  • Oxygen equilibrium curves using tonometry or specialized equipment like Hemox-Analyzer

  • Flash photolysis for kinetic association and dissociation rates of ligands

  • Differential scanning calorimetry for thermal stability

• Comparative analysis:

  • Side-by-side functional comparison with native D. akajei hemoglobin

  • Comparison with human HbA across multiple parameters

How do the structural differences between Dasyatis akajei HBB and human HBB influence oxygen binding properties?

The moderate reduction in oxygen affinity, haem-haem interaction, Bohr effect, and organic phosphate effect in D. akajei hemoglobin despite significant structural differences represents an intriguing evolutionary convergence of function . Key structural features influencing these properties include:

• Haem pocket architecture: Mutations and conformational changes around the haem in D. akajei HBB likely alter the microenvironment influencing oxygen association and dissociation kinetics. Research should focus on identifying specific residues that differ from human HBB and their effects on oxygen affinity.

• C-terminal region modifications: The C-terminal region of the beta subunit plays a crucial role in the allosteric transition between R (relaxed, high-affinity) and T (tense, low-affinity) states in human hemoglobin. The structural differences in this region in D. akajei HBB might contribute to the moderately reduced cooperativity observed .

• Interface interactions: The α1β2 interface forms critical contacts that stabilize the quaternary structure and transmit allosteric effects between subunits. Altered residue interactions at this interface in D. akajei hemoglobin would affect the communication between subunits during oxygen binding and release.

• Organic phosphate binding: The modified organic phosphate-binding site in D. akajei hemoglobin explains the reduced effect of organic phosphates on oxygen affinity. Detailed structural analysis of this region would elucidate the molecular basis for this physiological adaptation.

Methodological approaches to investigate these structure-function relationships should include:

• Site-directed mutagenesis to introduce human-like residues into recombinant D. akajei HBB

• Hybrid hemoglobins containing D. akajei and human subunits

• Molecular dynamics simulations to analyze the dynamics of key regions

• Hydrogen-deuterium exchange mass spectrometry to probe conformational flexibility

What protein engineering strategies can exploit unique properties of Dasyatis akajei HBB for improved oxygen carriers?

Recombinant hemoglobin-based oxygen carriers (HBOCs) development has focused on addressing key limitations including short vascular retention time, nitric oxide (NO) scavenging, and autooxidation. D. akajei HBB offers unique structural features that could be exploited through rational protein engineering:

• Genetic crosslinking approaches: Similar to the di-α-Gly linker used in rHb0.1 , genetic fusion of D. akajei alpha and beta subunits could generate stable tetramers resistant to dissociation. This approach might be particularly effective given the unique interface properties of D. akajei hemoglobin.

• NO scavenging reduction: Introducing mutations analogous to βV67W and αL29W that reduced NO scavenging 30-fold in human hemoglobin into the corresponding positions in D. akajei HBB might further reduce vasoactivity.

• Oxygen affinity modulation: The moderate oxygen affinity of D. akajei hemoglobin could be further optimized through targeted mutations informed by the structural differences from human HBB. Mutations similar to αH58Q that enhance oxygen dissociation from α-subunits could be adapted for D. akajei hemoglobin.

• Surface modification strategies: PEGylation and polymerization approaches used with human hemoglobin could be applied to engineered D. akajei hemoglobin to further extend vascular retention time and reduce vasoactivity.

A comprehensive engineering strategy might combine multiple approaches, as illustrated in this theoretical design:

What experimental approaches can assess the stability and autooxidation rates of recombinant Dasyatis akajei HBB?

Hemoglobin stability and resistance to oxidation are critical parameters for both basic research and potential applications. Comprehensive assessment requires multiple analytical techniques:

• Thermal stability assessment:

  • Differential scanning calorimetry (DSC) to determine melting temperatures (Tm)

  • Circular dichroism with thermal ramping to monitor secondary structure changes

  • Thermal challenge assays with subsequent analysis of functional properties

• Autooxidation kinetics:

  • Spectrophotometric monitoring of the conversion of oxy-hemoglobin (HbO₂) to met-hemoglobin (metHb) under controlled conditions

  • Measurement at multiple temperatures to determine activation energy

  • Experiments at varying pH values to assess pH-dependence of oxidation rates

  • Oxygen consumption measurements using oxygen electrodes

• Reactive oxygen species (ROS) generation:

  • Detection of superoxide using electron paramagnetic resonance (EPR) with spin traps

  • Hydrogen peroxide quantification using fluorescent or colorimetric assays

  • Analysis of protein oxidation markers (e.g., carbonyl content, dityrosine formation)

• Long-term stability studies:

  • Storage stability under various conditions (temperature, pH, buffer composition)

  • Periodic analysis of oligomeric state, metHb formation, and functional properties

  • Accelerated stability testing to predict long-term behavior

Results should be compared directly with both native D. akajei hemoglobin and human hemoglobin to evaluate relative stability and oxidation resistance. These comparisons would identify any unique properties of D. akajei HBB that might be advantageous for protein engineering applications.

How can computational modeling predict the effects of mutations in Dasyatis akajei HBB?

Computational modeling represents a powerful approach to guide experimental work on recombinant D. akajei HBB. Modern computational approaches can predict the effects of mutations and assist in rational design:

• Homology modeling: Using the high-resolution crystal structures of D. akajei hemoglobin as templates to model variants and mutants with high accuracy.

• Molecular dynamics (MD) simulations:

  • Analysis of protein dynamics in both oxy and deoxy states

  • Calculation of residue flexibility and conformational changes

  • Investigation of water and gas migration pathways

  • Prediction of the effects of mutations on structure and dynamics

• Quantum mechanics/molecular mechanics (QM/MM) approaches:

  • Detailed modeling of the heme environment and ligand interactions

  • Calculation of oxygen binding energetics

  • Prediction of redox properties and autooxidation propensity

• Free energy calculations:

  • Alchemical free energy methods to calculate mutation effects on stability

  • Potential of mean force calculations for ligand binding pathways

  • Binding free energy calculations for various ligands

• Machine learning approaches:

  • Prediction of mutation effects using trained models

  • Identification of non-obvious mutation sites for property optimization

  • Design of multiple mutations with synergistic effects

The computational workflow should include:

• Initial structure preparation and validation

• Systematic in silico mutagenesis of key residues

• Analysis of mutation effects on structure, dynamics, and predicted function

• Selection of promising mutations for experimental validation

• Iterative refinement based on experimental results

What methodological challenges exist in measuring the oxygen binding properties of recombinant Dasyatis akajei HBB?

Accurate measurement of oxygen binding properties presents several methodological challenges that researchers should address:

• Sample preparation considerations:

  • Ensuring consistent heme incorporation and oxidation state

  • Controlling buffer conditions (pH, ionic strength, temperature)

  • Removing any bound carbon monoxide from expression/purification

  • Maintaining protein stability during measurements

• Technical challenges in oxygen equilibrium curve (OEC) determination:

  • Selection of appropriate methodology (tonometry, thin-layer methods, Hemox-Analyzer)

  • Accurate control and measurement of oxygen partial pressure

  • Temperature control throughout measurements

  • Managing the effects of protein concentration on results

  • Accounting for autooxidation during measurements

• Data analysis complexities:

  • Fitting appropriate models (Hill equation, Adair equation, MWC model)

  • Determining P₅₀ (partial pressure of oxygen at 50% saturation) accurately

  • Calculating cooperativity coefficients (Hill coefficient)

  • Assessing the effects of allosteric effectors

• Special considerations for D. akajei HBB:

  • Potential differences in stability compared to mammalian hemoglobins

  • Species-specific effects of pH and temperature on oxygen binding

  • Unique responses to organic phosphates that might require modified protocols

Recommended methodology includes:

• Multiple independent measurements under identical conditions

• Validation using different techniques when possible

• Comprehensive controls including native D. akajei hemoglobin and human hemoglobin

• Careful attention to met-hemoglobin formation during experiments

How can CRISPR-Cas9 gene editing technologies be applied to study Dasyatis akajei HBB function?

The advances in CRISPR-Cas9 technology for human HBB gene correction can be adapted to study D. akajei HBB through both ex vivo and in vivo approaches:

• In vitro structure-function studies:

  • Precise genome editing to introduce specific mutations in recombinant expression systems

  • High-throughput creation of variant libraries for functional screening

  • Introduction of reporter tags without disrupting protein function

• Cell line engineering:

  • Creation of cell lines expressing wild-type and mutant D. akajei HBB

  • Development of reporter systems for hemoglobin assembly and function

  • Engineering of cells with humanized or chimeric hemoglobin for comparative studies

• Methodological considerations:

  • Design of highly specific guide RNAs targeting D. akajei HBB

  • Optimization of homology-directed repair templates for precise editing

  • Validation of editing efficiency using deep sequencing

  • Functional characterization of edited cells using spectroscopic and physiological assays

The CRISPR-Cas9 approach could employ high-fidelity Cas9 variants precomplexed with chemically modified guide RNAs similar to the methodology used for human HBB gene correction . This would minimize off-target effects while maximizing on-target editing efficiency.

What comparative analyses between human and Dasyatis akajei HBB can reveal evolutionary adaptations in oxygen transport?

Comparative analysis between these phylogenetically distant hemoglobins offers unique insights into evolutionary adaptation of oxygen transport proteins:

• Sequence-structure-function relationships:

  • Identification of conserved residues across >400 million years of evolution

  • Analysis of co-evolving residue networks essential for hemoglobin function

  • Mapping of species-specific adaptations to different physiological requirements

• Experimental approaches:

  • Reciprocal mutagenesis to introduce residues from each species into the other

  • Creation and functional testing of chimeric hemoglobins

  • Detailed kinetic analysis of oxygen association and dissociation rates

  • Comparative analysis of responses to allosteric effectors

• Physiological context analysis:

  • Correlation of hemoglobin properties with the environmental and physiological demands of stingrays vs. humans

  • Investigation of adaptations related to temperature sensitivity

  • Analysis of interactions with species-specific regulatory mechanisms

• Evolutionary rate analysis:

  • Calculation of substitution rates in different regions of the protein

  • Identification of sites under positive or purifying selection

  • Bayesian reconstruction of ancestral hemoglobin sequences

This comparative work would provide fundamental insights into hemoglobin evolution and the molecular basis of functional conservation despite significant sequence divergence between cartilaginous fish and mammals.