Recombinant Propithecus tattersalli Cytochrome c oxidase subunit 2 (MT-CO2)

What is Cytochrome c oxidase subunit 2 (MT-CO2) and why is it significant in P. tattersalli?

Cytochrome c oxidase subunit 2 (MT-CO2) is a protein encoded by the mitochondrial genome that plays a pivotal role in cellular respiration. In P. tattersalli and other mammals, MT-CO2 functions as a critical component of the cytochrome c oxidase complex, which performs a four-electron reduction of oxygen to form water during the final stage of electron transfer. This process creates the electrochemical gradient necessary for ATP synthesis .

MT-CO2 is particularly significant because it serves as the sole binding partner with cytochrome c and is the first recipient of its electrons. These electrons initially pass to the CuA site of cytochrome c oxidase, which is composed of two copper atoms coordinated with six ligands (two cysteines, two histidines, a methionine, and a peptide carbonyl of a glutamate), all contributed by MT-CO2 .

How does MT-CO2 in P. tattersalli differ from other lemur species?

Comparative genomic studies have shown that MT-CO2 exhibits heterogeneous rates of amino acid replacement among placental mammals, including lemurs. Unlike other mitochondrial genes that may evolve at more consistent rates, MT-CO2 shows variable evolutionary patterns that may reflect adaptations to specific ecological niches .

Genomic analyses of different Propithecus species, including P. tattersalli, P. diadema, P. coquereli, and P. verreauxi, have revealed distinct genetic variations. These species show different patterns in their genetic diversity, which could impact protein structure and function. P. tattersalli specifically shows genomic signatures that differentiate it from western sifaka species (P. coquereli and P. verreauxi) .

What are the primary research applications for recombinant P. tattersalli MT-CO2?

Recombinant P. tattersalli MT-CO2 is valuable for multiple research applications:

• Evolutionary studies: The protein serves as a molecular marker for investigating primate evolution, particularly the divergence and adaptation of lemur species in Madagascar .

• Structural biology research: The recombinant protein can be used to study the structural changes that have occurred in cytochrome c oxidase among different primate lineages .

• Functional assays: It enables the investigation of the altered physical interaction between cytochrome c oxidase and cytochrome c in primates .

• Immunological studies: The recombinant protein can be utilized in ELISA and other immunoassays to develop and validate detection methods .

• Conservation genomics: Studies of MT-CO2 can contribute to understanding the genetic diversity of this critically endangered species, supporting conservation efforts .

What experimental protocols are recommended for functional analysis of recombinant MT-CO2?

For functional analysis of recombinant P. tattersalli MT-CO2, the following protocols are recommended:

• Enzyme activity assays: Measure electron transfer rates using spectrophotometric methods that track the oxidation of reduced cytochrome c in the presence of the recombinant MT-CO2 integrated into artificial membrane systems.

• Protein-protein interaction studies: Employ techniques such as surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to quantify binding affinity between recombinant MT-CO2 and cytochrome c.

• Structural analysis: Use X-ray crystallography or cryo-electron microscopy to determine the three-dimensional structure of the protein and compare it with MT-CO2 from other species.

• SDS-PAGE analysis: Confirm protein purity and molecular weight. The recombinant protein typically shows greater than 90% purity as determined by SDS-PAGE .

• Reconstitution experiments: Incorporate the recombinant protein into liposomes or nanodiscs to study its function in a membrane-like environment.

How can researchers effectively utilize phylogenetic methods to study MT-CO2 evolution?

To effectively study MT-CO2 evolution using phylogenetic methods, researchers should:

• Sequence alignment and model selection: Align MT-CO2 sequences from multiple species and select appropriate evolutionary models. The Akaike Information Criterion (AIC) in programs like MrModeltest can be used to determine the best-fit model .

• Maximum likelihood and Bayesian analyses: Perform analyses using programs such as GARLI (for ML) and MrBayes (for Bayesian inference). For ML analyses, use the general time-reversible (GTR) model with rate heterogeneity (Γ) and a proportion of invariant sites (I) .

• Partitioned analysis framework: When analyzing MT-CO2 alongside other genes, implement a partitioned framework to allow independent parameter estimation for each locus .

• Bootstrap support assessment: Generate branch support for ML trees using bootstrapping with at least 100 pseudoreplicates .

• Bayesian concordance analysis: Assess concordance in gene trees across loci following established methods to identify potential discrepancies between gene and species trees .

• Demographic reconstruction: Use tools like the PSMC (Pairwise Sequentially Markovian Coalescent) method to infer changes in effective population size and population connectivity over time .

What are the optimal storage and handling conditions for recombinant P. tattersalli MT-CO2?

For optimal stability and activity of recombinant P. tattersalli MT-CO2, follow these storage and handling guidelines:

• Long-term storage: Store at -20°C or -80°C for extended periods. Aliquoting is necessary to prevent protein degradation from repeated freeze-thaw cycles .

• Working stocks: Store working aliquots at 4°C for up to one week to maintain activity .

• Storage buffer: The protein is typically supplied in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0 or a Tris-based buffer with 50% glycerol optimized for protein stability .

• Reconstitution protocol:

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

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

  • Add 5-50% glycerol (final concentration) and aliquot for long-term storage

• Avoid repeated freeze-thaw cycles: This is critical as repeated freezing and thawing significantly reduces protein activity and integrity .

What analytical techniques are most effective for characterizing recombinant MT-CO2 quality and function?

For comprehensive characterization of recombinant P. tattersalli MT-CO2, employ these analytical techniques:

• SDS-PAGE: Assess protein purity, which should be greater than 90% .

• Western blotting: Confirm protein identity using specific antibodies against either MT-CO2 or the His-tag.

• Mass spectrometry: Verify the exact molecular weight and identify any post-translational modifications or proteolytic degradation.

• Circular dichroism (CD) spectroscopy: Evaluate secondary structure integrity and thermal stability.

• Dynamic light scattering (DLS): Determine protein homogeneity and detect potential aggregation.

• Enzyme activity assays: Measure electron transfer rates and substrate binding affinities to confirm functional integrity.

• Protein-protein interaction assays: Verify binding to cytochrome c using co-immunoprecipitation, ELISA, or biophysical techniques like surface plasmon resonance.

How can researchers address challenges in expression and purification of recombinant MT-CO2?

To overcome common challenges in expression and purification of recombinant MT-CO2:

• Optimizing expression conditions:

  • Test multiple E. coli strains (BL21(DE3), Rosetta, etc.) to find optimal expression

  • Vary induction temperature (16-37°C) and IPTG concentration (0.1-1.0 mM)

  • Consider using auto-induction media for higher yields

  • Implement codon optimization for the P. tattersalli sequence to improve expression in E. coli

• Improving solubility:

  • Include solubility-enhancing tags (MBP, SUMO) in addition to the His-tag

  • Add detergents or lipids during lysis to stabilize this membrane protein

  • Consider expression as inclusion bodies followed by refolding protocols if active soluble protein is difficult to obtain

• Enhancing purification efficiency:

  • Optimize imidazole concentrations in binding and elution buffers

  • Implement a second purification step (ion exchange or size exclusion chromatography) to improve purity

  • Use mild detergents throughout purification to maintain native-like membrane protein folding

• Verifying functionality:

  • Develop activity assays specific to MT-CO2 function

  • Compare activity to predicted values based on native enzyme kinetics

  • Assess proper folding through limited proteolysis or tryptophan fluorescence

How can recombinant MT-CO2 contribute to understanding evolutionary adaptations in lemurs?

Recombinant MT-CO2 from P. tattersalli offers unique opportunities to investigate evolutionary adaptations in lemurs:

• Structural-functional relationship analysis: By comparing the structure and function of MT-CO2 across different lemur species, researchers can identify adaptive changes that correlate with ecological niches. The heterogeneous rates of amino acid replacement observed in MT-CO2 among placental mammals suggest possible adaptive evolution .

• Environmental adaptation markers: MT-CO2 variations may reflect adaptations to different environmental conditions. P. tattersalli inhabits dry deciduous and semi-evergreen forest fragments in northeastern Madagascar , and its MT-CO2 may show adaptations specific to this habitat.

• Metabolic efficiency assessment: Comparing enzyme kinetics of recombinant MT-CO2 from different Propithecus species can reveal adaptations for metabolic efficiency related to their leaf-based diets and energy requirements .

• Molecular clock applications: MT-CO2 sequence data can be used in molecular clock analyses to estimate divergence times between lemur species and reconstruct their evolutionary history in the context of Madagascar's geographic and climatic changes.

• Selection pressure analysis: Examining the ratio of non-synonymous to synonymous substitutions (dN/dS) in MT-CO2 across lemur species can identify regions under positive selection, potentially indicating functional adaptations .

What insights can comparative genomic analysis of MT-CO2 provide about P. tattersalli's conservation status?

Comparative genomic analysis of MT-CO2 can provide valuable insights for P. tattersalli conservation:

• Genetic diversity assessment: Genomic data from P. tattersalli shows distinct patterns in genetic diversity. The two sequenced P. tattersalli individuals showed approximately 22.5-22.7 million variants called after filtering, including about 20.7-20.9 million SNVs and 1.8 million indels .

• Population history reconstruction: Analysis of MT-CO2 and other genetic markers can reveal historical population bottlenecks and expansions. The genomes of all sifakas analyzed indicated cycles of increasing and declining inverse instantaneous coalescence rate (IICR) over the past million years, with pronounced decreases in the past 100,000 years, which may reflect changes in effective population size .

• Inbreeding detection: Homozygosity in MT-CO2 and other genetic regions can indicate inbreeding, which is concerning for endangered species like P. tattersalli.

• Adaptation potential evaluation: Genetic variants in functional genes like MT-CO2 can indicate a population's adaptive potential to changing environments, crucial for predicting responses to habitat alterations.

• Management unit definition: MT-CO2 variations across P. tattersalli populations can help define genetically distinct management units for conservation planning.

The table below summarizes key genomic metrics for P. tattersalli compared to other Propithecus species:

Data derived from comparative genomic analysis of sifakas .

How can functional studies of MT-CO2 inform our understanding of unique physiological adaptations in Golden-crowned Sifakas?

Functional studies of MT-CO2 can provide insights into the physiological adaptations of Golden-crowned Sifakas (P. tattersalli):

• Metabolic efficiency and dietary adaptations: P. tattersalli is primarily frugivorous and inhabits fragmented forest environments. Functional studies of MT-CO2 can reveal how its respiratory chain has adapted to support energy requirements from this diet. This is particularly relevant considering the genomic evidence of adaptation in digestion-related genes found in sifakas (although not specifically MT-CO2) .

• Thermal tolerance mechanisms: Golden-crowned sifakas live in dry deciduous forests with seasonal temperature variations. Analyzing the thermal stability and activity profiles of their MT-CO2 could reveal adaptations for maintaining mitochondrial function across temperature ranges.

• Activity pattern correlations: P. tattersalli is diurnal but differs from other sifakas in that it does not descend to the ground . This behavioral difference may correlate with specific energy metabolism adaptations that could be reflected in MT-CO2 function.

• Stress response mechanisms: Comparing the function of MT-CO2 under various stress conditions (oxidative, thermal, pH) can reveal adaptations that help these endangered lemurs cope with environmental challenges in their increasingly fragmented habitat.

• Comparison with captive populations: Activity patterns differ between wild and captive P. tattersalli, with captive individuals spending significantly more time resting and less time feeding than other sifaka species . Functional studies of MT-CO2 could help explain these metabolic differences.

What are the primary limitations when working with recombinant MT-CO2 rather than native protein?

Working with recombinant P. tattersalli MT-CO2 presents several methodological challenges:

• Structural differences: The recombinant protein, expressed in E. coli, lacks proper post-translational modifications that might be present in the native protein from mitochondria. This could affect structure, stability, and function.

• Membrane environment: MT-CO2 is a membrane protein that functions as part of a multi-subunit complex in the mitochondrial inner membrane. The recombinant protein lacks this native membrane environment, potentially affecting its folding and activity.

• Complex assembly challenges: In vivo, MT-CO2 functions as part of the larger cytochrome c oxidase complex with multiple subunits. The recombinant protein alone cannot fully recapitulate the native complex's function.

• Expression system limitations: E. coli expression systems may not produce protein with identical properties to that synthesized in eukaryotic mitochondria, potentially affecting folding, stability, and function.

• Functional assessment difficulties: Without the complete cytochrome c oxidase complex, assessing the true physiological function of isolated MT-CO2 is challenging and may not reflect in vivo activity.

What emerging technologies could advance research on P. tattersalli MT-CO2?

Several emerging technologies show promise for advancing P. tattersalli MT-CO2 research:

• AlphaFold and other AI protein structure prediction tools: These can provide detailed structural models of MT-CO2 and predict protein-protein interactions without requiring crystallization, which is challenging for membrane proteins.

• Nanodiscs and advanced membrane mimetics: These technologies can provide more native-like environments for reconstituting recombinant MT-CO2, enabling more physiologically relevant functional studies.

• Single-molecule techniques: Methods like single-molecule FRET can track conformational changes in MT-CO2 during its interaction with cytochrome c and electron transfer.

• CRISPR/Cas9 gene editing: This could enable the generation of cellular models expressing P. tattersalli MT-CO2, allowing studies in a more native context.

• Advanced mass spectrometry techniques: Native mass spectrometry and hydrogen-deuterium exchange mass spectrometry can provide insights into protein-protein interactions and dynamics of MT-CO2.

• In situ cryo-electron tomography: This technique could visualize the entire cytochrome c oxidase complex containing MT-CO2 in its native membrane environment.

• Long-read sequencing technologies: These can improve the genomic context of MT-CO2 in P. tattersalli, potentially revealing regulatory elements and heteroplasmies relevant to function.

How might MT-CO2 research contribute to broader conservation efforts for P. tattersalli?

MT-CO2 research has significant potential to contribute to P. tattersalli conservation:

• Non-invasive genetic monitoring: Developing MT-CO2-based markers could enable population monitoring from fecal samples, reducing the need to disturb wild populations.

• Adaptation potential assessment: Understanding the functional implications of MT-CO2 variants can help predict how different populations might respond to climate change and habitat alterations.

• Population viability modeling: MT-CO2 data can contribute to models predicting population responses to environmental changes, informing conservation priorities.

• Captive breeding program optimization: Genetic data including MT-CO2 can guide breeding programs to maintain genetic diversity, particularly important since P. tattersalli shows behavioral differences in captivity, including spending significantly more time resting than other captive sifaka species .

• Evolutionary importance documentation: MT-CO2 research highlights the unique evolutionary history of P. tattersalli, strengthening arguments for conservation based on their distinct genetic heritage and adaptation to northeastern Madagascar's forest fragments .

• Health monitoring protocols: Understanding normal MT-CO2 function can help develop markers for mitochondrial health assessment in both wild and captive populations, potentially detecting environmental stressors before they cause population decline.