Recombinant Liparis tunicatus Hemoglobin subunit beta-1 (hbb1)

What are the key structural features of Liparis tunicatus hemoglobin beta-1 subunit?

Liparis tunicatus hemoglobin beta-1 subunit contains several distinctive structural features. The beta chain displays an unusual substitution in NA2 (beta2) at the phosphate-binding site, and notably contains isoleucine at position E11 (beta67) instead of the valine typically found in other species. This substitution occurs at a critical position within the heme pocket that influences oxygen binding properties . In addition to these primary structural elements, the tertiary structure reveals a conformation that facilitates the formation of a ferric penta-coordinated species even at physiological pH, similar to some Antarctic fish hemoglobins . This structural arrangement plays a crucial role in determining the protein's functional characteristics and environmental adaptations.

How does the oxygen-binding affinity of Liparis tunicatus hemoglobin compare to other fish species?

Liparis tunicatus hemoglobin (specifically Hb 1, which is the major component) exhibits remarkably low oxygen affinity compared to many non-polar fish species. This characteristic is accompanied by pronounced Bohr and Root effects . The Bohr effect enables enhanced oxygen release in response to pH changes, while the Root effect allows for complete unloading of oxygen under specific physiological conditions. These properties differ significantly from hemoglobins found in temperate-water fish species but share similarities with some Antarctic notothenioids. The adaptive significance of these functional characteristics appears to be related to the cold, oxygen-rich polar marine environment.

A comparative analysis of oxygen affinity parameters is presented below:

Note: P₅₀ represents the partial pressure of oxygen at which the hemoglobin is 50% saturated; higher values indicate lower affinity.

What does the phylogenetic analysis of Liparis tunicatus hemoglobin reveal about evolutionary adaptations in polar fish?

Phylogenetic analysis indicates that Liparis tunicatus globins are evolutionarily close to the notothenioid clades, which aligns with established teleostean phylogenies . This evolutionary relationship provides important insights into molecular adaptations in polar environments. The close phylogenetic relationships between Cottoidei (to which L. tunicatus belongs) and Notothenioidei, combined with their similar life styles in polar waters, appear to be primary factors driving the evolution of their globin sequences .

Unlike Antarctic notothenioids that typically display minimal hemoglobin multiplicity, Arctic fish species often show greater hemoglobin diversity. While many Antarctic notothenioids have reduced the number of distinct hemoglobin types they express, possibly as a specialized adaptation to stable cold environments, L. tunicatus maintains a system with one major hemoglobin (Hb 1) and one minor component (Hb 2) . This pattern suggests different evolutionary pressures or adaptive strategies between Arctic and Antarctic fish species, potentially reflecting differences in their respective polar environments.

How does the amino acid composition of L. tunicatus hemoglobin beta-1 influence its cold adaptation mechanisms?

The amino acid composition of L. tunicatus hemoglobin beta-1 contains specific substitutions that contribute to its functionality in cold environments. The replacement of Val E11 (beta67) with Ile is particularly significant as this position interacts directly with the bound oxygen molecule . This substitution likely modifies the oxygen-binding pocket in a way that enables effective oxygen transport at near-freezing temperatures.

The cold adaptation mechanisms in L. tunicatus hemoglobin appear to work through multiple structural modifications:

• Altered heme pocket architecture that maintains functionality at low temperatures

• Amino acid substitutions that modify protein flexibility and stability

• Structural arrangements that permit enhanced oxygen release (via pronounced Bohr and Root effects) in cold, oxygen-rich environments

These adaptations differ from those found in some other cold-adapted proteins, which often show increased flexibility through decreases in hydrophobic core packing or reduced salt bridges. L. tunicatus hemoglobin demonstrates that cold adaptation can occur through various molecular mechanisms, including specialized modifications that affect ligand binding and release properties.

What are the optimal expression systems for producing recombinant L. tunicatus hemoglobin beta-1?

When expressing recombinant L. tunicatus hemoglobin beta-1, several expression systems can be considered based on current knowledge about recombinant hemoglobin production. While specific protocols for L. tunicatus hemoglobin expression are not extensively documented in the provided literature, comparable approaches used for other recombinant hemoglobins provide valuable methodological insights.

Escherichia coli expression systems have been successfully employed for various recombinant hemoglobins, as demonstrated with rHb (beta N108Q) . For L. tunicatus hemoglobin beta-1, the following methodological considerations are important:

• Expression vector selection: Vectors containing strong, inducible promoters (such as T7) are typically preferred

• Co-expression requirements: Successful expression often requires co-expression of both alpha and beta chains to obtain functional hemoglobin

• Heme incorporation: Supplementation with δ-aminolevulinic acid or hemin may be necessary to ensure proper heme incorporation

• Purification strategy: Affinity chromatography followed by ion-exchange chromatography has shown effectiveness for recombinant hemoglobins

When optimizing expression, researchers should monitor both yield and functional integrity, as improper folding or heme incorporation can compromise the distinctive oxygen-binding properties that make L. tunicatus hemoglobin scientifically valuable.

What spectroscopic methods are most effective for characterizing the structural properties of recombinant L. tunicatus hemoglobin beta-1?

Multiple spectroscopic approaches are valuable for characterizing recombinant L. tunicatus hemoglobin beta-1, each providing distinct but complementary structural information:

• Electron Paramagnetic Resonance (EPR) Spectroscopy: Particularly valuable for L. tunicatus hemoglobin analysis as it can detect the formation of ferric penta-coordinated species even at physiological pH, a characteristic feature of this hemoglobin . EPR provides critical information about the electronic structure and coordination state of the heme iron.

• Proton Nuclear Magnetic Resonance (¹H NMR): Essential for examining tertiary structure around the heme pockets and quaternary structural changes between different ligation states. This technique has been successfully applied to recombinant hemoglobins to detect structural similarities and differences compared to natural hemoglobins .

• UV-Visible Absorption Spectroscopy: Useful for monitoring the oxidation state of the heme iron (Fe²⁺ vs. Fe³⁺) and for tracking the kinetics of autoxidation, which is relevant given that L. tunicatus hemoglobin may exhibit distinctive autoxidation properties.

• Circular Dichroism (CD): Provides information about secondary structural elements and can be used to assess proper folding of the recombinant protein.

When implementing these methods, researchers should consider comparative analysis with other fish hemoglobins to contextualize the unique structural features of L. tunicatus hemoglobin beta-1.

How do mutations in key residues of recombinant L. tunicatus hemoglobin beta-1 affect oxygen-binding properties and structural stability?

Investigating the effects of strategic mutations in L. tunicatus hemoglobin beta-1 requires consideration of both the protein's unusual native substitutions and insights from other recombinant hemoglobin studies. Particularly relevant is the Val E11 (beta67) to Ile substitution in the native protein, which affects the heme pocket environment .

Studies on other recombinant hemoglobins have demonstrated that mutations at subunit interfaces and in the central cavity of the hemoglobin molecule can significantly alter oxygen-binding properties. For instance, the beta N108Q mutation in human hemoglobin resulted in low oxygen affinity, high cooperativity, enhanced Bohr effect, and slower autoxidation rates . Similarly, introducing mutations at position L29F in the alpha chain has shown stabilization against auto- and NO-induced oxidation .

When designing mutation studies for L. tunicatus hemoglobin beta-1, researchers should consider:

• Heme pocket residues that directly interact with the bound oxygen

• Residues at the alpha1-beta1 and alpha1-beta2 subunit interfaces that influence cooperativity

• The phosphate-binding site where L. tunicatus already displays an unusual substitution

• Residues that might influence the formation of the ferric penta-coordinated species

Experimental protocols should include comprehensive characterization of oxygen equilibrium curves under various conditions (pH, temperature, presence of allosteric effectors) and stability assessments through autoxidation rate measurements and thermal denaturation studies.

What methodologies are most effective for investigating the relationship between L. tunicatus hemoglobin structure and its adaptation to cold, high-pressure environments?

Investigating the relationship between L. tunicatus hemoglobin structure and environmental adaptations requires an integrated experimental approach:

• High-pressure spectroscopic studies: Utilizing specialized high-pressure cells with optical windows to perform absorption and fluorescence spectroscopy under varying pressure conditions that mimic the natural habitat of L. tunicatus.

• Temperature-dependent functional assays: Measuring oxygen binding properties (P₅₀, cooperativity, Bohr effect) across a temperature range (particularly at low temperatures) to quantify cold adaptation effects.

• Molecular dynamics simulations: Computational modeling of protein dynamics at various temperatures and pressures to identify flexibility/rigidity patterns and potential conformational changes associated with cold adaptation.

• Comparative structural analysis: High-resolution structural determination (X-ray crystallography or cryo-EM) of L. tunicatus hemoglobin in different ligation states, compared with hemoglobins from related temperate species.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): For analyzing protein flexibility and solvent accessibility at different temperatures, providing insights into cold-adaptation mechanisms.

The relationship between adaptation and evolutionary history should also be considered, as the close phylogenetic relationships between Cottoidei and Notothenioidei, combined with their similar lifestyles, appear to be driving factors in globin-sequence evolution . This evolutionary context provides a framework for understanding observed structural adaptations.

How can recombinant L. tunicatus hemoglobin beta-1 be utilized in creating hemoglobin-based oxygen carriers adapted for low-temperature applications?

Recombinant L. tunicatus hemoglobin beta-1 offers unique properties that could be valuable for developing specialized hemoglobin-based oxygen carriers (HBOCs) for low-temperature applications. The naturally low oxygen affinity, pronounced Bohr effect, and cold adaptation of this hemoglobin make it an interesting candidate for such applications .

Research approaches for developing low-temperature HBOCs using L. tunicatus hemoglobin should include:

• Protein engineering strategy: Creating chimeric proteins that combine the cold-adapted properties of L. tunicatus hemoglobin with stability features from other hemoglobins. Based on recombinant hemoglobin research, mutations such as those equivalent to alpha L29F could be introduced to enhance stability against autoxidation while maintaining low oxygen affinity .

• Surface modification protocols: Developing PEGylation or encapsulation methods optimized for low-temperature conditions to reduce immunogenicity and extend circulation time.

• Stability enhancement: Implementing cross-linking techniques that preserve the unique oxygen-binding properties while improving protein stability at various storage and application temperatures.

• Functional testing: Developing specialized assays to evaluate oxygen delivery efficiency under cold conditions that simulate potential application environments.

The design of such experiments should consider that recombinant hemoglobins with low oxygen affinity, high cooperativity, and stability against autoxidation (such as rHb beta N108Q) have been identified as potential candidates for HBOCs . L. tunicatus hemoglobin's natural adaptations to cold environments could provide additional advantages for specific low-temperature applications in biomedical research or tissue preservation.

What are the common challenges in purifying recombinant L. tunicatus hemoglobin beta-1 and how can they be addressed?

Purification of recombinant L. tunicatus hemoglobin beta-1 presents several challenges that researchers should anticipate and address methodically:

• Heme incorporation issues: Incomplete heme incorporation can result in heterogeneous protein preparations with variable functional properties. This can be addressed by optimizing heme supplementation during expression or through in vitro heme reconstitution protocols post-purification.

• Oxidation susceptibility: Hemoglobins are prone to oxidation during purification, potentially altering their functional characteristics. Maintaining reducing conditions throughout purification (using reducing agents like dithiothreitol or by working under nitrogen atmosphere) can mitigate this issue.

• Subunit dissociation: Hemoglobin tetramers can dissociate during purification, especially at low protein concentrations. Using stabilizing buffers containing specific ions or mild cross-linking may help maintain quaternary structure.

• Protein aggregation: The unique amino acid composition of L. tunicatus hemoglobin may lead to aggregation issues during concentration steps. This can be addressed by optimizing buffer conditions (pH, ionic strength, stabilizing additives) and using gentle concentration methods.

For quality control, researchers should implement multiple analytical methods to verify the integrity of purified protein:

• UV-visible spectroscopy to confirm proper heme incorporation (analyzing the Soret band and Q bands)

• Size-exclusion chromatography to assess quaternary structure integrity

• Functional assays to verify oxygen-binding properties

• Mass spectrometry to confirm primary sequence integrity

How should researchers interpret discrepancies between the functional properties of recombinant versus native L. tunicatus hemoglobin?

When discrepancies arise between recombinant and native L. tunicatus hemoglobin properties, systematic analysis is essential for accurate interpretation:

• Post-translational modification differences: Native L. tunicatus hemoglobin may contain post-translational modifications absent in recombinant versions. Mass spectrometry analysis can identify such modifications, allowing researchers to assess their functional impact.

• Expression system artifacts: Properties of recombinant proteins can be influenced by the expression system used. Comparing proteins expressed in different systems (bacterial, yeast, insect, mammalian) can help identify expression system-specific artifacts.

• Structural validation approaches: Discrepancies may reflect subtle structural differences. Comparative structural analysis using circular dichroism, NMR, or crystallography can help identify conformational variations between recombinant and native forms.

• Environmental context effects: Native hemoglobin functions within a specific cellular environment with various cofactors and allosteric regulators. Replicating these conditions in vitro for recombinant protein testing may reduce observed discrepancies.

When reporting such discrepancies, researchers should clearly document experimental conditions and consider evolutionary context. The unique adaptations of L. tunicatus hemoglobin to cold environments may make it particularly sensitive to experimental conditions , requiring carefully controlled comparative studies to accurately characterize both native and recombinant forms.

What emerging technologies could advance our understanding of L. tunicatus hemoglobin structure-function relationships?

Several cutting-edge technologies offer promising approaches for deeper insights into L. tunicatus hemoglobin:

• Cryo-electron microscopy (Cryo-EM): Recent advances in resolution capabilities make this technique valuable for examining hemoglobin conformational states without crystallization, potentially revealing dynamic aspects of cold adaptation mechanisms.

• Time-resolved X-ray crystallography: This approach could capture intermediate states during oxygen binding and release, providing unprecedented insights into the molecular basis of the pronounced Bohr and Root effects observed in L. tunicatus hemoglobin .

• Single-molecule FRET spectroscopy: By labeling specific residues, researchers could track real-time conformational changes during ligand binding under various temperature and pressure conditions, illuminating the molecular basis of cold adaptation.

• AlphaFold2 and related AI approaches: These computational methods could predict structural impacts of specific mutations or environmental conditions, guiding experimental design and interpretation of results.

• Nanopore technology: This emerging approach could potentially characterize single hemoglobin molecules, providing distribution information rather than ensemble averages and revealing heterogeneity in conformational states.

The application of these technologies to L. tunicatus hemoglobin could reveal fundamental insights into protein adaptation mechanisms in extreme environments and potentially inspire biomimetic approaches for protein engineering in biotechnological applications.

What are the key unanswered questions regarding the evolutionary adaptation of L. tunicatus hemoglobin to Arctic environments?

Despite significant advances in our understanding of L. tunicatus hemoglobin, several critical questions remain unanswered regarding its evolutionary adaptation:

• Selective pressures driving hemoglobin diversity: Why do Arctic fish like L. tunicatus display hemoglobin multiplicity patterns different from Antarctic notothenioids, despite both living in polar environments? This question addresses fundamental aspects of convergent versus divergent evolution in similar extreme environments .

• Functional significance of minor hemoglobin components: While the major hemoglobin component (Hb 1) has been characterized, the functional role of the minor component (Hb 2) in L. tunicatus remains unclear . Does it serve specialized physiological functions or represent evolutionary remnants?

• Molecular basis of simultaneous cold and pressure adaptation: The mechanisms by which L. tunicatus hemoglobin simultaneously adapts to both cold temperatures and pressure variations in its habitat remain incompletely understood.

• Comparative energetics of oxygen binding: How the thermodynamic parameters (enthalpy and entropy changes) associated with oxygen binding differ between L. tunicatus and temperate fish hemoglobins could provide insights into the energetic basis of cold adaptation.

• Evolutionary rate analysis: Whether the rate of molecular evolution in L. tunicatus hemoglobin genes differs from that of temperate relatives could reveal whether positive selection or genetic drift played the primary role in shaping its unique properties.

Addressing these questions will require integrated approaches combining phylogenetic analysis, structural biology, physiological studies, and ecological context to fully understand how evolutionary processes shaped these remarkable molecular adaptations to life in polar environments.