Recombinant Gorilla gorilla gorilla Hemoglobin subunit delta (HBD)
Description
Biological Role and Evolutionary Context
The δ-globin gene (HBD) encodes a minor component of adult hemoglobin (HbA2: α<sub>2</sub>δ<sub>2</sub>), which constitutes ~3% of total hemoglobin in primates . In Gorilla gorilla, HBD (UniProt ID: P61773) is retained as a functional gene, unlike in Old World Monkeys where it is pseudogenized . Evolutionary analysis reveals:
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Conservation: Gorilla HBD shares 98% sequence identity with human HBD, with key residues critical for heme binding and tetramer stability preserved .
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Developmental Regulation: Unlike humans, gorillas show delayed silencing of fetal γ-globin (HBG), but HBD expression remains low postnatally, similar to humans .
Evolutionary Constraints and Gene Conversion
Gorilla HBD exhibits unique evolutionary patterns:
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Gene Conversion: Limited gene conversion events with HBB compared to other primates, preserving functional motifs like GATA-1 (critical for fetal-to-adult hemoglobin switching) .
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Selection Pressures: dN/dS ratios (ω) suggest stronger purifying selection in apes (ω = 0.06) versus New/Old World Monkeys (ω = 0.43) .
Research Gaps and Future Directions
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Functional Studies: No in vivo data exist on recombinant gorilla HBD’s oxygen-carrying capacity or stability.
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Comparative Models: Cross-species expression in transgenic mice could elucidate its compensatory potential in hemoglobinopathies.
Product Specs
Q&A
Basic Research Questions
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What is Gorilla Hemoglobin subunit delta (HBD) and how does it differ from other hemoglobin subunits?
Gorilla Hemoglobin subunit delta (HBD) is one of several globin protein subunits comprising the hemoglobin molecule in western gorillas. Based on comparative genomics, HBD is part of the beta-type globin gene cluster arranged in the following order: 5'-epsilon (embryonic)-G gamma and A gamma (fetal)-psi beta (inactive)-delta and beta (adult)-3' . The delta subunit contributes to the formation of hemoglobin A2 (α2δ2), typically constituting about 2-3% of total adult hemoglobin.
The delta subunit differs structurally from the more abundant beta subunit by approximately 10 amino acids in humans, with likely similar patterns of variation in gorillas. These differences result in distinct biochemical properties affecting oxygen binding characteristics and responses to allosteric regulators. While delta hemoglobin has been less extensively studied than beta hemoglobin, analysis of delta-globin genes provides valuable insights into hemoglobinopathies and evolutionary adaptations in oxygen transport systems.
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What is the genomic organization of the beta-globin gene cluster in gorillas?
The beta-globin gene cluster in gorillas maintains the same organization pattern observed in humans and chimpanzees. According to comparative genomic studies, this cluster follows the arrangement: 5'-epsilon (embryonic)-G gamma and A gamma (fetal)-psi beta (inactive)-delta and beta (adult)-3' . This conservation indicates that the genes are organized according to their developmental expression timeline.
The presence of the pseudogene (ψβ) between fetal and adult genes represents a shared feature across these closely related primates. Research has shown that in gorillas, as in humans, each pseudogene shares substitutions in the initiator codon (ATG→GTA) and a substitution in codon 15 that generates a termination signal (TGG→) . This high degree of conservation in genomic organization suggests that the regulatory mechanisms controlling hemoglobin expression during development are similar across these primates, though specific sequence variations contribute to species-specific hemoglobin properties.
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What expression systems are most effective for producing recombinant gorilla HBD?
Based on established protocols for recombinant gorilla proteins, three primary expression systems have demonstrated effectiveness for producing functional hemoglobin subunits:
| Expression System | Advantages | Considerations | Application Suitability |
|---|---|---|---|
| E. coli | High yield, cost-effective, simplified purification | May require refolding, lacks post-translational modifications | Structural studies, antibody production |
| Yeast (P. pastoris, S. cerevisiae) | Eukaryotic processing, secretion capability, high yield | Longer production time, different glycosylation pattern | Functional studies requiring proper folding |
| Baculovirus/insect cells | Advanced eukaryotic processing, suitable for complex proteins | Higher cost, more complex methodology | Interaction studies, functional characterization |
For gorilla HBD specifically, E. coli systems have been used successfully for similar globin proteins when proper refolding protocols are employed . For studies investigating quaternary structure or protein-protein interactions, baculovirus expression systems may offer advantages due to their more sophisticated protein processing capabilities .
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What analytical methods are most reliable for confirming the identity and purity of recombinant gorilla HBD?
A comprehensive analytical workflow for recombinant gorilla HBD should include multiple orthogonal techniques:
| Analytical Technique | Purpose | Critical Parameters |
|---|---|---|
| SDS-PAGE | Size verification, initial purity assessment | Comparison with predicted molecular weight (~16 kDa) |
| Western blot | Identity confirmation | Antibody specificity, cross-reactivity control |
| Mass spectrometry (LC-MS/MS) | Sequence verification, post-translational modification analysis | Coverage percentage (>90% recommended) |
| Size exclusion chromatography | Oligomeric state determination, aggregation assessment | Calibration with appropriate standards |
| UV-vis spectroscopy | Heme incorporation analysis | Characteristic Soret band (~415 nm) and Q bands (500-600 nm) |
| Circular dichroism | Secondary structure confirmation | Alpha-helical content verification (~70% expected) |
For proper characterization, particular attention should be paid to heme incorporation analysis using UV-visible spectroscopy, as proper heme binding is critical for functional studies. Sequence verification through mass spectrometry is essential to confirm the absence of mutations or truncations that could affect functional studies.
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What purification strategies yield the highest purity recombinant gorilla HBD?
A systematic multi-step purification approach yields optimal results for recombinant gorilla HBD:
| Purification Step | Method | Key Parameters | Expected Outcome |
|---|---|---|---|
| Initial capture | Immobilized metal affinity chromatography (His-tag) | Imidazole gradient (20-500 mM) | >80% purity, removal of bulk contaminants |
| Intermediate purification | Ion exchange chromatography | pH 7.5-8.0 for anion exchange | >90% purity, separation from proteins with similar molecular weight |
| Polishing | Size exclusion chromatography | Flow rate <0.5 ml/min | >95% purity, removal of aggregates |
| Endotoxin removal | Polymyxin B columns | For E. coli-expressed protein | <0.5 EU/mg protein |
For functional studies requiring tetrameric hemoglobin, an additional reconstitution step with alpha-globin subunits would be necessary. The purification protocol should be validated by assessing protein homogeneity using analytical SEC, dynamic light scattering, and SDS-PAGE under both reducing and non-reducing conditions.
Advanced Research Questions
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How can evolutionary analysis of gorilla HBD inform our understanding of primate hemoglobin adaptation?
Evolutionary analysis of gorilla HBD offers valuable insights into hemoglobin adaptation through multiple methodological approaches:
| Analytical Approach | Methodology | Scientific Insights |
|---|---|---|
| Phylogenetic analysis | Maximum likelihood and Bayesian inference methods comparing delta-globin sequences across primates | Identification of positively selected sites potentially related to functional adaptations |
| Ancestral sequence reconstruction | Statistical inference of ancestral sequences, followed by recombinant expression | Direct measurement of functional shifts during evolutionary history |
| Molecular clock analysis | Dating sequence divergence events in delta globin | Correlation of adaptive changes with environmental or physiological shifts |
| Site-directed mutagenesis | Introducing gorilla-specific residues into human HBD or vice versa | Determination of functional effects of species-specific substitutions |
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What are the methodological challenges in characterizing allosteric regulation in recombinant gorilla HBD?
Investigating allosteric regulation in recombinant gorilla HBD presents several technical challenges requiring specialized methodological approaches:
| Challenge | Methodological Solution | Technical Considerations |
|---|---|---|
| Accurate tetramer reconstitution | Co-expression of alpha and delta chains or controlled in vitro assembly | Verification of stoichiometry by analytical ultracentrifugation |
| Heme incorporation efficiency | Optimized reconstitution protocols with ferrous hemin | Spectroscopic confirmation of correct heme coordination |
| Oxygen binding cooperativity measurement | Multi-wavelength spectroscopy under precisely controlled gas tensions | Temperature and pH control within ±0.1 units |
| Allosteric effector binding characterization | Isothermal titration calorimetry with varying effector concentrations | Background subtraction and control for buffer effects |
| Structural basis of allostery | X-ray crystallography in multiple allosteric states | Resolution ≤2.0 Å for meaningful mechanistic insights |
Studies in other hemoglobin systems demonstrate that allosteric effects can be highly context-dependent . The research on crocodilian hemoglobin revealed that gain of bicarbonate-sensitivity involved both direct effects of few replacements at key sites and indirect effects of numerous replacements at structurally disparate sites . Similar complexity is likely present in gorilla HBD, requiring comprehensive analysis of both direct binding interactions and conformational changes in distant regions of the protein.
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How does recombinant gorilla HBD compare functionally to other primate delta globins?
Comparative functional analysis of recombinant gorilla HBD with other primate delta globins requires systematic characterization across multiple parameters:
| Functional Parameter | Experimental Approach | Expected Species Differences |
|---|---|---|
| Oxygen affinity (P50) | Oxygen equilibrium curves at varying pH, temperature, and effector concentrations | 1-3 mmHg variation between closely related primates |
| Cooperativity coefficient (n50) | Hill plot analysis of oxygen binding curves | Subtle variations (±0.2) potentially reflecting different quaternary structures |
| Bohr effect magnitude | Oxygen affinity measurements across pH range 6.8-7.8 | Species-specific adaptations related to acid-base balance regulation |
| 2,3-BPG sensitivity | Oxygen binding with varying 2,3-BPG concentrations | Differences potentially reflecting adaptation to varying altitudinal ranges |
| Thermal stability | Differential scanning calorimetry | Correlation with species habitat temperature ranges |
Research approaches similar to those used in studying hemoglobin variants would allow identification of specific amino acid substitutions responsible for functional differences between gorilla and human HBD. Analyzing these differences in the context of each species' physiology and ecological niche can reveal adaptive significance of observed variations. Techniques for measuring these parameters must be standardized across species comparisons to ensure valid cross-species comparisons.
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What directed mutagenesis approaches are most effective for studying structure-function relationships in gorilla HBD?
Systematic directed mutagenesis of gorilla HBD enables detailed analysis of structure-function relationships through several specialized approaches:
| Mutagenesis Approach | Methodology | Research Application |
|---|---|---|
| Alanine scanning | Sequential replacement of charged/polar residues with alanine | Identification of functionally critical residues |
| Domain swapping | Exchange of entire structural domains between gorilla and human HBD | Localization of species-specific functional differences to particular regions |
| Ancestral state reconstruction | Reversion of gorilla-specific residues to ancestral states | Understanding evolutionary trajectory of functional properties |
| Charge reversal | Mutation of charged residues to opposite charge | Probing electrostatic contributions to structure and function |
| Introduction of spectroscopic probes | Cysteine substitution at key positions for fluorescence labeling | Monitoring conformational changes during allosteric transitions |
The research on crocodilian hemoglobin demonstrates that the effects of specific mutations can be highly context-dependent . When gain of bicarbonate-sensitivity was investigated, researchers found that "gain of bicarbonate-sensitivity involved direct effects of few replacements at key sites in combination with indirect effects of numerous replacements at structurally disparate sites" . This suggests that combinatorial mutagenesis approaches, rather than single-site mutations, may be necessary to fully understand structure-function relationships in gorilla HBD.
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What analytical techniques provide the most comprehensive characterization of oxygen binding properties in recombinant gorilla HBD?
A multi-technique approach yields the most comprehensive characterization of oxygen binding properties in recombinant gorilla HBD:
| Analytical Technique | Measured Parameters | Technical Requirements |
|---|---|---|
| Multi-wavelength spectroscopy | Complete oxygen equilibrium curves, P50, n50 | Temperature-controlled sample chamber, precise gas mixing |
| Stopped-flow spectroscopy | Association (kon) and dissociation (koff) rate constants | Dead time <2ms, photodiode array detection |
| Resonance Raman spectroscopy | Heme pocket structural changes upon oxygenation | Laser excitation at heme absorption bands |
| Hydrogen-deuterium exchange mass spectrometry | Regional dynamics and solvent accessibility changes | Ultra-high performance liquid chromatography, high-resolution mass spectrometry |
| X-ray crystallography | Atomic structure in different ligation states | Crystals of both oxy and deoxy forms at ≤2.0 Å resolution |
| Molecular dynamics simulations | Conformational dynamics and allosteric pathways | Validated force fields, microsecond simulation timescales |
These techniques should be applied under physiologically relevant conditions, including appropriate pH (7.2-7.4), temperature (37°C), and ion concentrations. The integration of data from multiple techniques allows construction of comprehensive models explaining the relationship between structure, dynamics, and oxygen binding function in gorilla HBD.
Unique Aspects of Gorilla HBD Research
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How does the Delta Hemoglobin (ΔHb) concept apply to research with recombinant gorilla HBD?
While Delta Hemoglobin (ΔHb) typically refers to the difference between preoperative and postoperative hemoglobin levels in clinical settings , this concept has analogous applications in recombinant gorilla HBD research:
| Clinical ΔHb Concept | Analogous Application in Gorilla HBD Research |
|---|---|
| Change in hemoglobin level | Quantifiable changes in HBD stability under varying conditions |
| Threshold for transfusion | Critical concentrations for functional studies |
| Association with clinical outcomes | Correlation of stability parameters with evolutionary fitness |
Research shows that in clinical settings, "larger ΔHb values, as well as receipt of transfusion, were strongly associated with risk of perioperative complication" . In parallel, experimental protocols for recombinant gorilla HBD should monitor protein stability and concentration changes during functional assays, as significant degradation or precipitation could affect interpretation of results. Standardized methods for quantifying and reporting these changes would enhance reproducibility across research groups.
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What are the unique challenges in studying potential deletions or mutations in gorilla HBD?
Investigation of deletions or mutations in gorilla HBD presents several specialized challenges:
| Challenge | Methodological Approach | Technical Considerations |
|---|---|---|
| Identification of naturally occurring variants | Genomic sequencing across multiple gorilla populations | Sample collection ethics, conservation implications |
| Characterization of deletion effects | PCR-based approaches similar to those used for hemoglobinopathies | Primer design accounting for gorilla-specific sequences |
| Modeling functional impact of variants | Recombinant expression of identified variants | Comparison with wild-type protein under identical conditions |
| Detection of regulatory region mutations | Promoter-reporter assays | Species-appropriate cellular context |
Studies of hemoglobin deletions in other contexts, such as the Southeast Asian δβ° deletion , provide methodological frameworks adaptable to gorilla HBD research. These studies employed Southern analysis with restriction enzymes like XbaI, AvaII, and EcoRI, and PCR amplification strategies targeting specific fragments . Similar approaches could be applied to characterize any naturally occurring variants in gorilla populations, providing insights into both evolutionary history and potential functional adaptations.
| Deletion Detection Method | Application to Gorilla HBD | Expected Results |
|---|---|---|
| Southern blot analysis | Genomic DNA digested with specific restriction enzymes | Fragment size differences indicating deletions |
| PCR amplification of deletion junctions | Using primers spanning potential breakpoints | Amplification products specific to deletion variants |
| Sequencing of junction fragments | Sanger or next-generation sequencing | Precise mapping of deletion breakpoints |
These approaches would allow comprehensive characterization of any structural variations in the gorilla HBD gene, contributing to our understanding of hemoglobin evolution in great apes.
