Recombinant Nycticebus coucang Cytochrome c oxidase subunit 2 (MT-CO2)

How does MT-CO2 from Nycticebus coucang compare structurally to that of other mammals?

The MT-CO2 protein in Nycticebus coucang shows significant sequence homology with other mammals, reflecting its conserved function in cellular respiration. Comparative analyses reveal:

Compared to other loris species, molecular phylogenetic studies have shown that MT-CO2 can be used as a genetic marker to distinguish between different Nycticebus species and populations, providing insight into their evolutionary relationships .

What are the recommended protocols for expressing and purifying recombinant Nycticebus coucang MT-CO2?

For successful expression and purification of recombinant Nycticebus coucang MT-CO2, researchers should follow these methodological steps:

• Vector Selection and Cloning:

  • Isolate the MT-CO2 gene from Nycticebus coucang mitochondrial DNA

  • Clone the full coding region into an appropriate expression vector

  • Verify the sequence integrity through DNA sequencing to confirm the presence of start (ATG) and stop codons

• Expression System Selection:

  • E. coli expression systems are commonly used for recombinant mitochondrial proteins

  • Consider using specialized strains designed for expression of membrane-associated proteins

• Protein Expression:

  • Culture transformed cells under optimized conditions

  • Induce protein expression with appropriate inducers

  • Monitor expression levels via SDS-PAGE

• Purification Strategy:

  • Lyse cells using appropriate buffer systems containing glycerol and reducing agents

  • Purify using affinity chromatography if a tag system is employed

  • Consider employing ion exchange chromatography as a secondary purification step

  • Use gel filtration for final polishing and buffer exchange

• Storage:

  • Store in buffer containing 50% glycerol at −20°C for extended storage

  • For highest stability, consider storage at −80°C

  • Avoid repeated freeze-thaw cycles

A typical formulation for storage would include Tris-based buffer (pH 8.0), 0.1M NaCl, 50% glycerol, and 2mM DTT .

What are the most effective approaches for studying protein-protein interactions involving MT-CO2?

Several methodological approaches have proven effective for studying protein-protein interactions involving MT-CO2:

• Co-Immunoprecipitation (Co-IP):

  • Utilize antibodies specific to MT-CO2 or potential interaction partners

  • For MT-CO2, consider rabbit recombinant monoclonal antibodies that have shown specificity for cytochrome c oxidase subunits in other species

  • Analyze precipitates using Western blotting or mass spectrometry

• Crosslinking Mass Spectrometry:

  • Apply chemical crosslinkers to stabilize transient interactions

  • Digest crosslinked proteins and analyze fragments by MS/MS

  • This approach is particularly valuable for mapping interaction interfaces

• Yeast Two-Hybrid or Bacterial Two-Hybrid Assays:

  • Construct fusion proteins with DNA-binding and activation domains

  • Screen for interactions through reporter gene activation

  • Note that membrane proteins like MT-CO2 may require modified approaches, such as split-ubiquitin systems

• Bioluminescence Resonance Energy Transfer (BRET) or Fluorescence Resonance Energy Transfer (FRET):

  • Tag MT-CO2 and potential partners with appropriate fluorescent proteins

  • Monitor energy transfer as indication of physical proximity

  • Particularly useful for dynamic, real-time interaction studies

• Surface Plasmon Resonance (SPR):

  • Immobilize purified MT-CO2 on a sensor chip

  • Measure binding kinetics with potential interaction partners

When designing experiments to study interactions between MT-CO2 and cytochrome c, researchers should consider the conserved acidic amino acid residues (two Asp and two Glu) that are likely involved in these interactions .

How does MT-CO2 sequence analysis contribute to understanding the evolutionary relationships among loris species?

MT-CO2 sequence analysis has been instrumental in elucidating evolutionary relationships among loris species, particularly within the genus Nycticebus:

• Phylogenetic Groupings:

  • Molecular phylogenetic analyses reveal that Nycticebus species form distinct evolutionary lineages

  • MT-CO2 sequences help differentiate between species and populations that may be morphologically similar

• Divergence Time Estimation:

  • Molecular clock analyses using MT-CO2 sequences suggest that:

    • The divergence between Xanthonycticebus (pygmy lorises) and Nycticebus occurred approximately 6 million years ago

    • N. coucang from Sumatra diverged from the Java 2 clade approximately 1.08 million years ago (HPD: 1.90–0.46 mya)

    • The MRCA (Most Recent Common Ancestor) of Sumatran N. coucang populations is estimated at 0.33 million years ago (HPD: 0.47–0.05 mya)

• Geographic Structuring:

  • MT-CO2 sequences reveal significant structuring between different geographic populations:

    • N. coucang from peninsular Malaysia are paraphyletic to Sumatran N. coucang samples

    • Peninsular Malaysian N. coucang (mostly N. c. malayanus) form a distinct clade from N. c. tennaserimensis

• Species Delineation:

  • Analysis of MT-CO2 has supported the recognition of several distinct species and subspecies within the slow loris complex

  • The most recent common ancestor (MRCA) of the N. coucang (Sumatra) is estimated at 0.33 million years ago, indicating relatively recent diversification

Researchers should note that MT-CO2, as a mitochondrial gene, is maternally inherited and should be used in conjunction with nuclear markers for comprehensive phylogenetic analysis to account for potential issues such as introgression or incomplete lineage sorting.

What evidence exists for natural selection or adaptive evolution in MT-CO2 among different loris populations?

Several lines of evidence suggest selective pressures and adaptive evolution have shaped MT-CO2 variation among loris populations:

• Conservation of Functional Domains:

  • Critical functional regions, such as the copper binding site (involving two Cys and two His residues), show strong conservation across species, indicating purifying selection on functionally essential residues

  • The region containing aromatic residues involved in electron transfer also shows high conservation

• Population-Specific Variation:

  • Distinct MT-CO2 sequence variants correspond to geographic distribution of Nycticebus populations

  • This suggests potential adaptation to different environmental conditions or ecological niches

• Molecular Evolution Rates:

  • Studies on mitochondrial genes suggest variable evolutionary rates between different loris lineages

  • This pattern could reflect different selective pressures or bottleneck events in population history

• Protein Structural Implications:

  • Mutations in MT-CO2 may affect protein structure and function, potentially leading to changes in energy metabolism

  • Some variants may confer advantages in different ecological settings or dietary regimes

• Dietary Adaptation:

  • The diet of Nycticebus coucang is specialized, and studies of captive slow lorises indicate distinct nutrient requirements and digestive capabilities

  • Changes in MT-CO2 might correlate with metabolic adaptations to different diets or energy-use patterns across populations

When investigating selection on MT-CO2, researchers should consider:

• Calculating dN/dS ratios to measure selection pressure

• Performing McDonald-Kreitman tests to evaluate evidence of adaptive evolution

• Mapping variations onto protein structural models to assess functional implications of amino acid substitutions

How can recombinant MT-CO2 be used to study mitochondrial disorders and their potential treatments?

Recombinant Nycticebus coucang MT-CO2 offers valuable research applications for studying mitochondrial disorders:

• Functional Complementation Studies:

  • Express recombinant Nycticebus coucang MT-CO2 in cell lines with defective human MT-CO2

  • Assess whether the loris protein can restore respiratory function

  • This approach can help understand functional conservation and divergence

• Structure-Function Relationship Analysis:

  • Create chimeric proteins combining domains from human and loris MT-CO2

  • Introduce specific mutations found in human patients into the recombinant loris protein

  • Analyze how these mutations affect protein stability, assembly, and function

• Drug Screening Platforms:

  • Develop high-throughput assays using recombinant MT-CO2 to screen compounds that might:

    • Stabilize mutant proteins

    • Enhance cytochrome c oxidase assembly

    • Modulate enzyme activity

  • Compare effects across species to understand evolutionary conservation of drug responses

• Disease-Associated Mutation Analysis:

  • Pathogenic mutations in human MT-CO2 have been identified, such as:

    • M29K (T7671A) mutation associated with mitochondrial myopathy that reduces O₂ consumption to 25% of control values

    • Q111Frameshift mutation associated with Leigh-like syndrome that drastically reduces complex activity to as low as 11-19% of normal levels

  • Introducing equivalent mutations into recombinant loris MT-CO2 could help understand their structural and functional impacts

• Protein-Protein Interaction Studies:

  • Investigate how disease-causing mutations affect interactions with other respiratory complex subunits

  • Study assembly intermediates and factors that could enhance proper complex formation

Researchers should note that differences between species may affect translational applicability, necessitating careful experimental design and controls when applying findings from loris MT-CO2 to human mitochondrial disorders.

What roles does MT-CO2 play in climate change research and CO₂ monitoring studies?

While MT-CO2 (the protein) and CO₂ (the gas) are distinct entities, there are interesting research connections between them:

• Evolutionary Adaptation to Changing Environments:

  • MT-CO2 sequence evolution in different species might reflect adaptation to changing atmospheric oxygen and CO₂ levels over evolutionary time

  • Comparing MT-CO2 sequences across species from different ecological niches could provide insights into respiratory adaptations to various oxygen/CO₂ levels

• Respiratory Efficiency and Carbon Footprint:

  • MT-CO2 function directly affects cellular respiratory efficiency

  • Understanding the relationship between MT-CO2 structure, function, and energetic efficiency could inform biomimetic approaches to reducing carbon emissions

• Biomonitoring Applications:

  • The function of cytochrome c oxidase can be affected by environmental pollutants

  • MT-CO2 activity assays could potentially serve as biomarkers for environmental stress in wildlife

• Methodological Parallels with Atmospheric CO₂ Monitoring:

  • While not directly related to the protein, it's worth noting the methodological sophistication in atmospheric CO₂ monitoring techniques:

    • The World Meteorological Organization (WMO) Global Atmosphere Watch coordinates high-quality greenhouse gas observations globally

    • Different methodologies like WDCGG, NOAA, and GFIT analyze CO₂ measurements with varying approaches

    • Networks with continental sites improve early detection of changes in biogenic emissions

• Carbon Capture Technologies:

  • Industrial processes for atmospheric CO₂ capture implement chemical loops that parallel biological pathways

  • Research into MT-CO2 function could potentially inspire biomimetic approaches to carbon capture

Understanding the function of MT-CO2 across species provides contextual knowledge for interpreting the broader biological aspects of carbon cycling and adaptation to changing atmospheric conditions.

What are the common challenges in isolating authentic MT-CO2 sequences and avoiding nuclear mitochondrial DNA contamination?

Researchers face several methodological challenges when working with MT-CO2 sequences:

• Nuclear Mitochondrial DNA (NUMT) Contamination:

  • Fragments of mitochondrial DNA can be incorporated into the nuclear genome and become pseudogenes

  • NUMTs can be as large as 5-8kb and are more common than initially assumed

  • Studies have found that the transfer of mitochondrial DNA to the nuclear genome can occur through multiple independent transposition events

• Identification of NUMTs:

  • Recently transferred NUMTs may retain high sequence identity to mitochondrial counterparts

  • NUMTs typically evolve 10-39 times slower than authentic mitochondrial DNA, particularly at the 3rd position of protein-coding genes

  • They may contain frameshift mutations or premature stop codons, though recent transfers might not

• PCR Amplification Biases:

  • Universal primers can preferentially amplify nuclear copies due to their more conserved nature

  • PCR may amplify NUMTs even when using purified mitochondrial extracts

• Strategies to Avoid NUMT Contamination:

  Strategy	Advantages	Limitations
  Use of mitochondria-rich tissues	Higher ratio of mtDNA to nuclear DNA	Cannot completely eliminate NUMTs
  Long-range PCR (9kb fragments)	Exceeds typical NUMT size	Technical difficulty with degraded samples
  RT-PCR from mRNA	Ensures transcribed mitochondrial genes	RNA instability; limited to short fragments
  Species-specific primers	Reduces chance of NUMT amplification	Requires prior sequence knowledge
  Phylogenetic screening	Identifies divergent sequences	Post-sequencing analysis required

• Authentication Methods:

  • Comparison with complete mitochondrial genome references

  • Relative rate tests to identify slowly evolving sequences (potential NUMTs)

  • Checking for reading frame integrity in protein-coding genes like MT-CO2

Researchers should implement multiple strategies, with long-range PCR of larger fragments being particularly effective for avoiding NUMT contamination when working with Nycticebus coucang MT-CO2.

What are the optimal conditions for maintaining stability and activity of recombinant MT-CO2 in experimental applications?

Maintaining the stability and activity of recombinant MT-CO2 requires careful attention to several factors:

• Buffer Composition:

  • Optimal buffer: Tris-based buffer (pH 8.0) containing:

    • 0.1M NaCl for ionic strength

    • 50% glycerol as a stabilizing agent

    • 2mM DTT as a reducing agent to protect sulfhydryl groups

• Storage Considerations:

  • Store at -20°C for routine use

  • For extended storage, maintain at -80°C

  • Avoid repeated freeze-thaw cycles by preparing working aliquots

  • Working aliquots can be kept at 4°C for up to one week

• Protein Concentration Effects:

  • Maintain protein at concentrations of ~1mg/ml to reduce aggregation

  • Higher concentrations may be used if additional stabilizing agents are included

• Oxidation Prevention:

  • MT-CO2 contains cysteine residues critical for copper binding and function

  • Maintain reducing conditions with DTT or β-mercaptoethanol

  • Consider argon or nitrogen overlay for long-term storage

• Factors Affecting Experimental Performance:

  Factor	Optimal Condition	Effect on Activity
  pH	7.5-8.0	Maximum enzyme stability
  Temperature	4°C (storage), 20-25°C (experiments)	Lower temperatures reduce denaturation
  Salt concentration	0.1-0.2M NaCl	Maintains proper folding
  Detergents	Low concentrations of mild detergents	May help stability of this membrane-associated protein
  Metal ions	Avoid chelating agents	Preserve copper cofactors essential for function

• Activity Preservation:

  • Addition of phospholipids may help maintain native-like environment

  • Consider measuring activity immediately after thawing for consistent results

  • For functional studies, reconstitution in liposomes may better preserve activity

• Aggregation Prevention:

  • Filter solutions before storage

  • Centrifuge samples briefly before use to remove any aggregates

  • Consider addition of low concentrations of non-ionic detergents for particularly challenging preparations

Following these guidelines will help maintain the structural integrity and functional activity of recombinant Nycticebus coucang MT-CO2 during storage and experimental applications .

How do feeding ecology studies of slow lorises inform our understanding of MT-CO2 function and evolution?

The relationship between feeding ecology and MT-CO2 evolution in slow lorises presents a fascinating research area:

• Dietary Specialization and Metabolic Adaptation:

  • Slow lorises have specialized dietary requirements

  • Diet 2 (based on wild feeding ecology) and Diet 3 (formulated to target wild-type nutrient content) show more similarity to each other than to conventional captive diets

  • These dietary adaptations may correlate with specific MT-CO2 sequence variations that optimize metabolic efficiency for processing natural food sources

• Digestive Efficiency Parameters:

  • Studies on captive Nycticebus species show specific digestive parameters:

  Parameter	Diet 1 (Original)	Diet 2 (Naturalistic)	Diet 3 (Recommended)
  Transit Time	Variable	Closer to wild estimates	Intermediate
  Mean Retention Time	Generally longer	More efficient	Optimized
  Crude Protein Digestibility	Lower	Higher	Optimized
  Fiber Digestibility	Limited	Enhanced	Enhanced
  Calcium Digestibility	Suboptimal	Improved	Improved

  These digestive parameters may reflect adaptations in energy metabolism pathways where MT-CO2 plays a crucial role

• Energy Expenditure in Wild Individuals:

  • Studies calculating nutrient intake rates and energy expenditure in wild lorises provide insights into metabolic demands

  • These energetic requirements likely shaped the evolution of efficient mitochondrial function, including MT-CO2 optimization

• Species-Specific Variations:

  • Different Nycticebus species (N. javanicus, N. coucang, N. menagensis) show variations in digestive efficiency

  • These variations may correlate with subtle differences in MT-CO2 sequence and function that optimize energy extraction for specific dietary niches

• Methodological Implications:

  • When studying MT-CO2 function in vitro, researchers should consider replicating physiologically relevant conditions that match the species' natural metabolic environment

  • Experimental designs should account for the specialized metabolic adaptations of slow lorises when interpreting MT-CO2 functional data

Understanding the feeding ecology of Nycticebus coucang provides critical context for interpreting the functional significance of MT-CO2 variations and their role in metabolic adaptation to specific ecological niches .

What are the most promising applications of recombinant MT-CO2 in biotechnology and biomedical research?

Recombinant Nycticebus coucang MT-CO2 offers several promising applications in biotechnology and biomedical research:

• Bioenergetics Research:

  • Serves as a model system for studying fundamental aspects of mitochondrial electron transport

  • Enables comparison of respiratory chain efficiency across species

  • Provides insights into evolutionary adaptations in energy metabolism

• Biomedical Applications:

  • Structural templates for designing drugs targeting human cytochrome c oxidase disorders

  • Development of therapies for mitochondrial diseases involving MT-CO2 mutations

  • Model for studying how specific mutations affect enzyme activity and stability

• Protein Engineering:

  • Creation of chimeric proteins with enhanced stability or activity

  • Development of biosensors for detecting environmental toxins that affect respiratory function

  • Design of biocatalysts for oxygen reduction reactions

• Immunological Tools:

  • Production of specific antibodies for mitochondrial research

  • Development of immunoassays for detecting mitochondrial dysfunction

  • Comparison with human MT-CO2 for understanding species-specific immune responses

• Diagnostic Applications:

  • Development of assays to detect mitochondrial dysfunction

  • Design of screening platforms for mitochondrial toxicity of drugs or environmental agents

  • Creation of reference standards for clinical laboratories studying mitochondrial disorders

• Phylogenetic and Conservation Applications:

  • Molecular markers for species identification in wildlife forensics

  • Tools for assessing genetic diversity in endangered loris populations

  • Models for studying adaptation to environmental change

• Biomimetic Technology:

  • Inspiration for designing efficient oxygen reduction catalysts

  • Models for developing artificial photosynthetic systems

  • Templates for creating biomimetic membranes with controlled proton transport properties

The unique evolutionary position of Nycticebus coucang and its specialized metabolism make its MT-CO2 particularly valuable for comparative studies that bridge fundamental research and applied biotechnology.