Recombinant Gazella spekei Cytochrome c oxidase subunit 2 (MT-CO2)

What is the basic structure and function of Gazella spekei Cytochrome c oxidase subunit 2?

Gazella spekei Cytochrome c oxidase subunit 2 (MT-CO2) is a transmembrane protein component of Complex IV in the mitochondrial respiratory chain. It contains 227 amino acids with the sequence: MAYPMQLGFQDATSPIMEELLHFHDHTLMIVFLISSLVLYVISLMLTTKLTHTST MDAQE VETIWTILPAIILILIALPSLRILYMMDEINNPSLTVKTMGHQWYWSYEYTDYEDLSFDS YMIPTSELKPGELRLLEVDNRVVLPMEMTIRMLISSEDVLHSWAVPSLGLKTDAI PGRLN QTTLMSARPGLYYGQCSEICGSNHSFMPIVLELVPLKYFEKWSASML .

Functionally, Cytochrome c oxidase subunit 2 plays a vital role in cellular respiration by receiving electrons from cytochrome c and transferring them to the cytochrome a3-CuB binuclear center. As part of the larger Complex IV, it contributes to oxygen reduction and proton transport across the mitochondrial membrane, which is essential for ATP synthesis .

How does the metabolic profile of Gazella spekei relate to its Cytochrome c oxidase activity?

The metabolic profile of Gazella spekei shows uniquely high resting metabolic rates (RMR) compared to other gazelle species. With an RMR of 432 ± 32 kJ kg BM^-0.75 d^-1, G. spekei demonstrates approximately 47% higher metabolic rate than would be predicted by standard models (Kleiber ratio: 1.47 ± 0.11) . This elevated metabolic activity correlates with respiratory chain function, where cytochrome c oxidase plays a crucial role.

Notably, G. spekei has the lowest respiratory quotient (RQ = 0.76 ± 0.02) among compared gazelle species, indicating a greater reliance on fat metabolism over carbohydrates . This metabolic adaptation may reflect evolutionary pressure in its arid native habitat and potentially affects the regulation of cytochrome c oxidase activity in mitochondrial respiration.

What are the key differences between Gazella spekei MT-CO2 and corresponding proteins in other gazelle species?

When comparing Gazella spekei MT-CO2 with other gazelle species, several notable differences emerge in both sequence conservation and physiological context:

These physiological differences suggest potential adaptive variations in MT-CO2 function or regulation across gazelle species . While complete comparative sequence analysis is not provided in the available data, the elevated metabolic rate in G. spekei correlates with its adaptation to its ecological niche and may reflect differences in electron transport chain efficiency.

What are the optimal storage and handling conditions for Recombinant Gazella spekei MT-CO2?

For optimal storage and handling of Recombinant Gazella spekei MT-CO2:

• Store the protein at -20°C for routine storage

• For extended storage stability, maintain at -80°C

• Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

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

• The shelf life in liquid form is typically 6 months at -20°C/-80°C

• Lyophilized preparations maintain stability for approximately 12 months at -20°C/-80°C

When designing experiments, consider preparing single-use aliquots during initial thawing to maintain protein integrity throughout the research timeline. Buffer composition should be verified as compatible with downstream applications, as the standard storage buffer contains Tris-based components with 50% glycerol .

How should researchers design expression systems for producing Recombinant Gazella spekei MT-CO2?

Designing effective expression systems for Recombinant Gazella spekei MT-CO2 requires careful consideration of several factors:

• Expression Vector Selection: The commercially available protein utilizes an E. coli expression system with an N-terminal 10xHis-tag . This approach balances yield with proper protein folding.

• Codon Optimization: As G. spekei is not a model organism, codon optimization for the expression host is critical. The full-length protein (227 amino acids) may contain rare codons that require optimization.

• Tag Placement Considerations: The N-terminal His-tag configuration minimizes interference with the transmembrane domains. Alternative tagging strategies should avoid disrupting the protein's functional domains.

• Expression Conditions: Transmembrane proteins often require specialized expression conditions. Lowering induction temperature (16-20°C) and using milder inducers can improve proper folding.

• Verification Methods: Confirm successful expression using Western blotting with anti-His antibodies and verify structural integrity through circular dichroism spectroscopy.

For researchers developing their own expression systems, it's advisable to compare results with the commercially available standard (CSB-CF015073GBB) to validate their methods .

What are the key considerations for designing experiments to study MT-CO2 activity in comparative physiology?

When designing experiments to study MT-CO2 activity in comparative physiology:

• Metabolic Rate Measurements: Use respirometry chambers equipped with O₂/CO₂ analyzers as employed in gazelle species studies. Ensure adequate acclimation periods (minimum 1 hour) before recording data to minimize stress effects .

• Environmental Controls: Standardize temperature, humidity, and light conditions across species comparisons. Gazelles show temporal fluctuations in O₂ consumption with decreases toward night's end .

• Normalization Approaches: Express metabolic data per unit metabolic body mass (BM^-0.75) for valid cross-species comparisons. This allows proper contextualization of MT-CO2 function across body size differences .

• Diet Standardization: Provide consistent nutrition (e.g., lucerne ad libitum) during experimental periods to control for dietary influences on respiratory quotient.

• Data Collection Duration: Record measurements over complete diurnal cycles (minimum 24 hours) to capture the full range of metabolic variability.

For isolation of mitochondria and direct measurement of cytochrome c oxidase activity, tissues should be processed immediately after collection, with activity assays performed at physiologically relevant temperatures specific to the species being studied.

How can Recombinant Gazella spekei MT-CO2 be utilized in evolutionary biology studies?

Recombinant Gazella spekei MT-CO2 offers several valuable applications in evolutionary biology research:

• Molecular Clock Analyses: MT-CO2, being mitochondrially encoded, evolves at a relatively consistent rate. Researchers can use sequence data to estimate divergence times between Gazella species and other bovids. The full-length sequence (amino acids 1-227) provides sufficient data points for robust phylogenetic analyses .

• Adaptive Evolution Studies: The elevated metabolic rate of G. spekei (47% higher than predicted by Kleiber's law) suggests positive selection on respiratory chain components. Researchers can employ Ka/Ks ratio analyses to identify positively selected sites in MT-CO2 that may contribute to this metabolic adaptation.

• Structure-Function Relationship: Using the recombinant protein with site-directed mutagenesis, researchers can investigate how specific amino acid changes influence electron transfer efficiency. This approach can reveal the molecular basis for metabolic adaptations in arid-adapted gazelle species.

• Comparative Functional Assays: By comparing the kinetic properties of MT-CO2 across gazelle species (G. spekei, G. gazella, N. soemmerringii), researchers can correlate functional differences with the documented metabolic variations (RMR/BMR ratios of 1.47, 1.27, and 1.06 respectively) .

These applications can provide insights into how selection pressures in different ecological niches drive the evolution of energy metabolism at the molecular level.

What methodological approaches should be used when investigating the relationship between MT-CO2 structure and metabolic adaptation?

Investigating the relationship between MT-CO2 structure and metabolic adaptation requires an integrated approach:

• Structural Biology Methods:

  • X-ray crystallography or cryo-EM to determine the three-dimensional structure of G. spekei MT-CO2

  • Molecular dynamics simulations to model protein-protein interactions within Complex IV

  • Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions potentially involved in adaptive functions

• Functional Assays:

  • Oxygen consumption measurements using recombinant proteins in reconstituted systems

  • Electron transfer kinetics compared across species with varying metabolic rates

  • Proton pumping efficiency measurements to correlate structure with bioenergetic output

• Integrative Analysis:

  • Correlation of specific structural features with metabolic parameters (e.g., RMR values of 432 ± 32, 371 ± 31, and 310 ± 32 kJ kg BM^-0.75 d^-1 for G. spekei, G. gazella, and N. soemmerringii respectively)

  • Computational modeling of electron flow through mutant variants to predict adaptive advantages

• Ecological Context Integration:

  • Connect molecular findings with ecological parameters (temperature, aridity, food availability)

  • Correlate structural adaptations with behavioral energy conservation strategies

This multidisciplinary approach allows researchers to establish causative relationships between specific amino acid variants in MT-CO2 and the observed metabolic phenotypes across gazelle species.

How can researchers effectively design experiments to examine the post-translational modifications of Gazella spekei MT-CO2?

Examining post-translational modifications (PTMs) of Gazella spekei MT-CO2 requires a systematic experimental design:

• PTM Identification Strategy:

  • High-resolution mass spectrometry (MS) using multiple fragmentation techniques (CID, ETD, HCD)

  • Enrichment approaches for specific modifications (phosphopeptide enrichment, glycopeptide capture)

  • Targeted MS/MS analysis focusing on the conserved histidine-tyrosine cross-link (equivalent to bovine His-240/Tyr-244)

• Comparative PTM Analysis:

  • Compare PTM patterns between recombinant protein (expressed in E. coli) and native protein isolated from G. spekei tissue

  • Analyze differences in PTM profiles between G. spekei and other gazelle species with different metabolic profiles

• Functional Significance Assessment:

  • Site-directed mutagenesis of potential PTM sites

  • Activity assays comparing wild-type and mutant proteins

  • Structural analysis to determine how PTMs affect protein conformation and interaction surfaces

• Physiological Context Experiments:

  • Investigate how environmental factors (temperature, pH, oxidative stress) affect PTM patterns

  • Correlate changes in PTM state with functional parameters like oxygen affinity or electron transfer rates

Researchers should be aware that the recombinant protein expressed in E. coli may lack mammalian-specific PTMs, necessitating comparison with native protein for comprehensive PTM analysis.

What are common experimental challenges when working with Recombinant Gazella spekei MT-CO2 and how can they be addressed?

Researchers commonly encounter several challenges when working with Recombinant Gazella spekei MT-CO2:

• Protein Aggregation Issues:

  • Challenge: As a transmembrane protein, MT-CO2 has hydrophobic regions prone to aggregation.

  • Solution: Add 0.1-0.5% mild detergents (DDM, CHAPS) to buffers; maintain glycerol concentration at 20-50%; perform experiments at 4°C whenever possible .

• Activity Loss During Storage:

  • Challenge: Functional decline despite proper storage conditions.

  • Solution: Prepare single-use aliquots; verify protein integrity by SDS-PAGE before experiments; consider adding reducing agents (1-2mM DTT) if disulfide formation is suspected .

• Inconsistent Experimental Results:

  • Challenge: Variable activity measurements between experiments.

  • Solution: Standardize protein concentration determination methods; use internal controls; ensure consistent metal ion concentrations (copper and iron) in buffers as these are essential for MT-CO2 function .

• Recombinant vs. Native Protein Differences:

  • Challenge: E. coli-expressed protein may lack post-translational modifications found in native MT-CO2.

  • Solution: Compare key functional parameters with native protein when possible; acknowledge limitations in results interpretation.

• Electron Transfer Measurement Difficulties:

  • Challenge: Complex kinetics of electron transfer are difficult to measure accurately.

  • Solution: Use stopped-flow spectroscopy with defined electron donors; calibrate systems with well-characterized cytochrome c oxidase preparations.

Maintaining detailed experimental records of protein batch, storage duration, and handling conditions can help identify sources of variability in research outcomes.

How should researchers interpret metabolic data in relation to MT-CO2 function across different gazelle species?

When interpreting metabolic data in relation to MT-CO2 function across gazelle species, researchers should consider several key factors:

• Scaling Relationships and Allometry:

  • Metabolic rates should be normalized to metabolic body mass (BM^-0.75) to account for size differences between species (G. spekei: 12.4kg; G. gazella: 16.3kg; N. soemmerringii: 35.1kg) .

  • Use both standard scaling equations (Kleiber and McNab) to calculate expected values versus observed measurements.

• Respiratory Quotient (RQ) Interpretation:

  • The lower RQ of G. spekei (0.76 ± 0.02) compared to N. soemmerringii (0.82 ± 0.03) indicates different substrate utilization patterns that may influence MT-CO2 function .

  • Consider substrate availability effects on enzyme kinetics when comparing across species.

• Environmental Adaptation Context:

  • The higher RMR/BMR ratio in G. spekei (1.47 ± 0.11) compared to G. gazella (1.27 ± 0.11) and N. soemmerringii (1.06 ± 0.11) suggests adaptive differences in mitochondrial function .

  • Correlate differences in MT-CO2 sequence or activity with habitat-specific demands.

• Temporal Variation Assessment:

  • Account for circadian patterns in metabolism, as all gazelle species showed temporal fluctuations in O₂ consumption and CO₂ production .

  • Design sampling protocols that capture these variations for accurate comparisons.

• Statistical Approach:

  • Apply appropriate statistical tests (ANOVA with Tukey's HSD) for between-species comparisons as demonstrated in previous gazelle metabolism studies .

  • Report effect sizes alongside p-values to quantify the magnitude of differences.

When directly connecting metabolic parameters to MT-CO2 function, researchers should establish clear mechanistic links rather than assuming correlation implies causation.

What validation approaches should be employed to ensure the authenticity and activity of Recombinant Gazella spekei MT-CO2?

To ensure the authenticity and activity of Recombinant Gazella spekei MT-CO2, researchers should implement a comprehensive validation strategy:

• Structural Validation:

  • Sequence Verification: Confirm the expressed protein matches the expected sequence (MAYPMQLGFQDATSPIMEELLHFHDHTLMIVFLISSLVLYVISLMLTTKLTHTST MDAQE VETIWTILPAIILILIALPSLRILYMMDEINNPSLTVKTMGHQWYWSYEYTDYEDLSFDS YMIPTSELKPGELRLLEVDNRVVLPMEMTIRMLISSEDVLHSWAVPSLGLKTDAI PGRLN QTTLMSARPGLYYGQCSEICGSNHSFMPIVLELVPLKYFEKWSASML) using mass spectrometry .

  • Size Confirmation: Verify molecular weight by SDS-PAGE and western blotting with anti-His antibodies to detect the N-terminal 10xHis tag .

  • Folding Assessment: Employ circular dichroism spectroscopy to confirm secondary structure elements characteristic of cytochrome c oxidase subunit 2.

• Functional Validation:

  • Electron Transfer Activity: Measure the rate of electron transfer from reduced cytochrome c using spectrophotometric assays.

  • Oxygen Consumption: Quantify oxygen reduction rates in reconstituted systems or membrane preparations.

  • Copper Content Analysis: Verify the presence of copper cofactors essential for MT-CO2 function using atomic absorption spectroscopy or inductively coupled plasma mass spectrometry.

• Comparative Benchmarking:

  • Compare activity metrics with other commercially available cytochrome c oxidase preparations.

  • Establish relative activity compared to native protein from G. spekei tissue when available.

  • Develop specific activity measurements (activity units per mg protein) as quality control benchmarks.

• Stability Assessment:

  • Monitor activity retention over time under different storage conditions.

  • Evaluate thermal stability using differential scanning fluorimetry.

  • Determine pH stability profile to inform buffer selection for downstream applications.

These validation approaches provide a robust framework to ensure that experimental outcomes truly reflect the biological properties of Gazella spekei MT-CO2 rather than artifacts of recombinant expression or storage conditions.

What emerging technologies could enhance our understanding of Gazella spekei MT-CO2 structure-function relationships?

Several cutting-edge technologies show promise for advancing our understanding of Gazella spekei MT-CO2:

• Cryo-Electron Microscopy (Cryo-EM):

  • Enables visualization of MT-CO2 within the entire Complex IV assembly at near-atomic resolution

  • Allows study of conformational changes during the catalytic cycle without crystallization constraints

  • Particularly valuable for membrane proteins like MT-CO2 that are challenging to crystallize

• AlphaFold2 and Deep Learning Approaches:

  • Accurate prediction of protein structures can complement experimental data

  • Allows modeling of species-specific variations in MT-CO2 structure even without direct crystallographic data

  • Facilitates in silico mutagenesis studies to predict functional consequences of amino acid substitutions

• Single-Molecule Techniques:

  • Single-molecule FRET to track conformational changes during electron transfer

  • Optical tweezers combined with electrical recordings to correlate mechanical changes with proton pumping

  • These approaches can reveal heterogeneity in molecular behavior masked in ensemble measurements

• In-cell Structural Biology:

  • NMR and EPR techniques adapted for in-cell measurements

  • Provides structural information in the native cellular environment

  • Can reveal physiologically relevant protein-protein interactions

• Multi-omics Integration:

  • Combining proteomics, metabolomics, and transcriptomics data

  • Creates a systems-level understanding of how MT-CO2 variations impact cellular metabolism

  • Particularly relevant for understanding the elevated metabolic rate observed in G. spekei

Integrating these technologies will provide unprecedented insights into how the unique properties of G. spekei MT-CO2 contribute to its distinctive metabolic profile.

How might climate change impact the metabolic adaptations reflected in Gazella spekei MT-CO2 function?

Climate change poses significant challenges to the metabolic adaptations reflected in Gazella spekei MT-CO2 function:

• Temperature Adaptation Mechanisms:

  • G. spekei's elevated metabolic rate (RMR/BMR ratio of 1.47 ± 0.11) may represent an adaptation to its current climate niche

  • Rising temperatures could shift the optimal efficiency range of MT-CO2 electron transfer

  • Research should investigate temperature-dependent kinetics of the recombinant protein under projected climate scenarios

• Water Conservation and Metabolic Function:

  • The low respiratory quotient (RQ = 0.76 ± 0.02) of G. spekei indicates fat metabolism preference

  • Increased aridity may further constrain water availability, potentially selecting for even more efficient MT-CO2 variants

  • Experimental designs should examine how dehydration affects MT-CO2 activity in tissue samples

• Food Resource Alterations:

  • Changing plant communities will affect nutrient availability and quality

  • Substrate availability influences electron transport chain function

  • Metabolic chamber studies with varying nutrient compositions can model these effects

• Population Genetic Implications:

  • Climate-driven range contractions may reduce genetic diversity in MT-CO2 variants

  • Decreased adaptive potential could limit response to changing conditions

  • Conservation genetics approaches should monitor MT-CO2 sequence diversity in wild populations

These research directions can provide critical insights for conservation planning while advancing our fundamental understanding of metabolic adaptation mechanisms at the molecular level in changing environments.

What interdisciplinary approaches could maximize the research value of Recombinant Gazella spekei MT-CO2?

Maximizing the research value of Recombinant Gazella spekei MT-CO2 requires innovative interdisciplinary approaches:

• Evolutionary Physiology Integration:

  • Combine molecular evolution analyses with physiological measurements

  • Map sequence variations to metabolic phenotypes across multiple gazelle species

  • Correlate selection signatures with ecological parameters in G. spekei's habitat

• Bioengineering Applications:

  • Use G. spekei MT-CO2 as a template for designing more efficient bioenergetic systems

  • Explore biomimetic applications based on its high-efficiency electron transfer (as suggested by elevated RMR)

  • Develop synthetic biology approaches incorporating advantageous features from G. spekei MT-CO2

• Computational Biology and Machine Learning:

  • Develop predictive models of protein function based on sequence data

  • Use machine learning to identify subtle patterns in electron transfer efficiency

  • Create virtual screening platforms to identify molecules that modulate MT-CO2 activity

• Conservation Genomics Connections:

  • Link MT-CO2 genetic diversity to population viability in endangered gazelle species

  • Develop MT-CO2 haplotype markers for monitoring wild populations

  • Use functional genomics to assess adaptive potential under climate change

• Comparative Medicine Applications:

  • Investigate how G. spekei's efficient MT-CO2 might inform research on human mitochondrial disorders

  • Explore therapeutic relevance of specific structural features for conditions involving cytochrome c oxidase deficiency

  • Develop experimental models incorporating G. spekei MT-CO2 variants to test therapies

By bridging these diverse disciplines, researchers can extract maximum scientific value from studies of this specialized protein while contributing to both basic science knowledge and applied solutions to contemporary challenges.