Recombinant Monosiga brevicollis SURF1-like protein (18583)

What is Monosiga brevicollis and why is it significant for evolutionary studies?

Monosiga brevicollis is a marine choanoflagellate that was selected for genome sequencing due to its laboratory cultivation ease, availability in monoxenic culture, and thorough consideration in phylogenetic studies. As one of the closest living relatives of animals, M. brevicollis provides a foundation for reconstructing the early evolutionary history of multicellular animals and for interpreting the physiology of these ecologically significant organisms . Its genome sequence, published in Nature in 2008, has become an essential reference for comparative genomics studies examining the transition from unicellular to multicellular life forms .

What is the SURF1-like protein in Monosiga brevicollis?

The SURF1-like protein in Monosiga brevicollis (gene ID: 18583) is a full-length protein comprising 261 amino acids. It shares homology with the human SURF1 protein, which functions in the assembly or maintenance of cytochrome c oxidase (COX) complexes . The recombinant protein is typically produced with an N-terminal 10xHis-tag in E. coli expression systems . The protein's amino acid sequence exhibits characteristic transmembrane domains similar to those found in human SURF1, suggesting conservation of structural features across evolutionary distance .

How is recombinant Monosiga brevicollis SURF1-like protein (18583) typically produced?

The recombinant M. brevicollis SURF1-like protein is typically produced in an in vitro E. coli expression system . The protein is expressed with an N-terminal 10xHis-tag to facilitate purification. Following expression, the protein is purified and provided either in liquid form or as a lyophilized powder. The liquid formulation is generally suspended in Tris/PBS-based buffer with 6% trehalose at pH 8.0 . The expression system is optimized to produce the full-length protein spanning amino acids 1-261, preserving the protein's structural integrity for downstream applications.

What are the optimal storage conditions for recombinant SURF1-like protein?

For optimal stability, recombinant Monosiga brevicollis SURF1-like protein should be stored at -20°C to -80°C upon receipt, with aliquoting necessary for multiple use scenarios to avoid repeated freeze-thaw cycles . The shelf life varies depending on storage conditions, buffer components, storage temperature, and the intrinsic stability of the protein itself. Generally, the liquid form maintains stability for approximately 6 months at -20°C to -80°C, while the lyophilized form can be stable for up to 12 months under the same conditions . For working solutions, short-term storage at 4°C is recommended, but this should be limited to minimize degradation.

How can researchers validate the functional activity of recombinant SURF1-like protein?

Validating functional activity of recombinant SURF1-like protein requires multiple complementary approaches:

• Structural Integrity Assessment: Using circular dichroism spectroscopy to confirm proper protein folding.

• Membrane Integration Assays: Based on methods used for human SURF1, researchers can assess whether the protein properly integrates into membranes through:

  • Alkaline carbonate extraction resistance tests

  • Proteinase K digestion susceptibility in artificial membrane vesicles

• Functional Complementation: Testing whether the M. brevicollis SURF1-like protein can rescue COX activity in SURF1-deficient cells, similar to complementation assays performed with human SURF1 .

• Protein-Protein Interaction Studies: Co-immunoprecipitation assays to identify binding partners within the mitochondrial membrane, particularly focusing on interactions with cytochrome c oxidase assembly factors.

What experimental controls should be included when studying SURF1-like protein function?

When designing experiments to study SURF1-like protein function, researchers should include the following controls:

• Negative Controls:

  • Empty vector controls in expression systems

  • Truncated protein variants lacking key functional domains

  • Heat-denatured protein samples

• Positive Controls:

  • Human SURF1 protein in parallel experiments

  • Known functional homologs from closely related species

• Specificity Controls:

  • Mutant variants with altered transmembrane domains

  • Co-expression of N- and C-terminal transmembrane domains as independent entities

• System Controls:

  • SURF1-deficient cell lines with established phenotypes

  • Wild-type M. brevicollis cells for comparative analyses

These controls help distinguish specific protein functions from experimental artifacts and provide benchmarks for interpreting results from functional assays.

How can M. brevicollis SURF1-like protein be used to study the evolution of mitochondrial assembly factors?

The M. brevicollis SURF1-like protein provides a unique opportunity to study the evolution of mitochondrial assembly factors:

What insights can M. brevicollis SURF1-like protein provide into pre-metazoan mitochondrial function?

The study of M. brevicollis SURF1-like protein offers critical insights into pre-metazoan mitochondrial function:

• Assembly Mechanism Conservation: By comparing the interaction of SURF1-like protein with other mitochondrial proteins in M. brevicollis, researchers can determine which aspects of COX assembly evolved prior to the emergence of animals.

• Metabolic Adaptation: Investigating whether the SURF1-like protein in M. brevicollis responds differently to environmental stressors compared to animal SURF1 may reveal how mitochondrial assembly processes adapted during the evolution of multicellularity.

• Regulatory Network Analysis: Examining the transcriptional regulation of SURF1-like protein in M. brevicollis compared to animals can identify when complex regulatory networks governing mitochondrial biogenesis emerged.

• Alternative Functions: The SURF1-like protein in M. brevicollis may perform additional functions not seen in animal orthologs, potentially revealing ancestral roles that were lost during specialization.

How does the domain structure of M. brevicollis SURF1-like protein compare to its human counterpart?

The domain structure comparison between M. brevicollis SURF1-like protein and human SURF1 reveals both conservation and divergence:

How can the M. brevicollis model system be used to study SURF1-like protein function?

Monosiga brevicollis has emerged as a valuable model system for studying the function of proteins like SURF1 in a pre-metazoan context:

• Laboratory Cultivation: M. brevicollis can be easily grown in the laboratory and is available in monoxenic culture, making it amenable to experimental manipulation .

• Genetic Modification: Recent advances have established methods for genetic manipulation in M. brevicollis, allowing researchers to:

  • Create knockout or knockdown strains for SURF1-like protein

  • Introduce tagged versions of the protein for localization studies

  • Generate point mutations to test functional hypotheses

• Stress Response Studies: M. brevicollis responds to pathogenic bacteria like Pseudomonas aeruginosa, providing a system to study how mitochondrial proteins like SURF1 respond to cellular stress .

• Live Imaging: The unicellular nature of M. brevicollis facilitates live imaging of mitochondrial dynamics and protein localization, allowing researchers to visualize SURF1-like protein behavior in real-time.

How does bacterial pathogenicity affect mitochondrial proteins like SURF1 in M. brevicollis?

Research has shown that M. brevicollis is susceptible to bacterial pathogens like Pseudomonas aeruginosa, which induces cell death . This interaction provides an opportunity to study how mitochondrial assembly factors respond to pathogenic stress:

• Stress-Induced Regulation: Exposure of M. brevicollis to P. aeruginosa likely triggers stress responses that could alter SURF1-like protein expression or localization. Time-course experiments measuring SURF1-like protein levels during bacterial challenge could reveal regulatory mechanisms.

• Mitochondrial Integrity: P. aeruginosa infection causes cell death in M. brevicollis , potentially through disruption of mitochondrial function. Investigating whether SURF1-like protein levels or activity are specifically targeted during infection could reveal pathogenic mechanisms.

• Evolutionary Comparison: Comparing how SURF1-like protein responds to bacterial challenge in M. brevicollis versus animal cells may illuminate the evolution of mitochondrial stress responses.

• Protective Mechanisms: Determining whether overexpression of SURF1-like protein can confer protection against P. aeruginosa-induced cell death would provide insights into potential protective roles beyond COX assembly.

What can be learned about human SURF1-related diseases by studying the M. brevicollis ortholog?

The study of M. brevicollis SURF1-like protein offers valuable perspectives on human SURF1-related diseases such as Leigh syndrome:

• Functional Conservation: If the M. brevicollis SURF1-like protein can rescue SURF1-deficient human cells, this would indicate functional conservation of critical domains involved in disease pathogenesis. This would help identify the core functional elements that, when mutated, lead to disease .

• Novel Functional Domains: The M. brevicollis protein may contain domains or structural elements not recognized in human SURF1 that contribute to function. These could represent overlooked therapeutic targets for SURF1-related diseases.

• Compensatory Mechanisms: Choanoflagellates like M. brevicollis may possess redundant pathways that compensate for SURF1 dysfunction, potentially revealing alternative approaches to treating SURF1 deficiency in humans.

• Evolutionary Medicine Insights: Understanding how SURF1 function has been conserved or modified through evolution may explain why SURF1 mutations primarily affect the nervous system in humans despite ubiquitous expression .

What experimental approaches can determine if recombinant SURF1-like protein can complement human SURF1 deficiency?

To determine whether M. brevicollis SURF1-like protein can complement human SURF1 deficiency, researchers can employ the following experimental approaches:

• Expression in Patient Fibroblasts: Transfecting SURF1-deficient patient fibroblasts with M. brevicollis SURF1-like protein constructs and measuring:

  • COX activity restoration using enzymatic assays

  • Assembly of COX complex using blue native PAGE

  • Steady-state levels of both nuclear- and mitochondrial-encoded COX subunits

• Mitochondrial Localization Verification: Confirming proper mitochondrial targeting using:

  • Epitope (FLAG)-tagged M. brevicollis SURF1-like protein expression in human cells

  • Mitochondrial import assays to verify membrane potential-dependent import and processing

  • Subcellular fractionation and immunoblotting

• Domain Swapping Experiments: Creating chimeric proteins containing domains from both human SURF1 and M. brevicollis SURF1-like protein to identify which regions are functionally interchangeable and which are species-specific.

• Rescue of Cellular Phenotypes: Assessing whether expression of M. brevicollis SURF1-like protein can rescue cellular phenotypes in SURF1-deficient cells, such as:

  • Mitochondrial membrane potential defects

  • Increased reactive oxygen species production

  • Altered mitochondrial morphology

What are the future research directions for M. brevicollis SURF1-like protein studies?

Future research on M. brevicollis SURF1-like protein should focus on several promising directions:

• Structural Biology: Determining the three-dimensional structure of the M. brevicollis SURF1-like protein would provide invaluable insights into functional domains and evolutionary conservation.

• Protein Interaction Networks: Comprehensive identification of binding partners in M. brevicollis compared to metazoans would illuminate the evolution of mitochondrial protein complexes.

• Functional Diversity: Investigating whether SURF1-like proteins in choanoflagellates have functions beyond COX assembly that were lost during animal evolution.

• Environmental Adaptation: Studying how SURF1-like protein function responds to changing environmental conditions in M. brevicollis may reveal adaptive mechanisms relevant to mitochondrial disease.

• Therapeutic Applications: Exploring whether unique features of the M. brevicollis SURF1-like protein could be exploited for therapeutic approaches to human SURF1-related diseases.

• Integration with Immune Function: Given the established role of M. brevicollis as a model for studying immune responses , investigating potential interactions between SURF1-like protein function and pre-metazoan immunity could reveal unexpected connections between mitochondrial function and early immune system evolution.

How can structural biology techniques be applied to study M. brevicollis SURF1-like protein?

Structural biology techniques offer powerful approaches to understanding the M. brevicollis SURF1-like protein:

• Cryo-Electron Microscopy: This technique can determine the structure of membrane-embedded SURF1-like protein, potentially in complex with interacting partners.

• X-ray Crystallography: While challenging for membrane proteins, this approach could resolve atomic-level details of soluble domains or the full protein in detergent micelles.

• NMR Spectroscopy: Solution NMR could characterize dynamic regions and identify conformational changes upon substrate binding.

• Molecular Dynamics Simulations: Using the amino acid sequence as input, researchers can model the M. brevicollis SURF1-like protein structure and simulate its behavior in a membrane environment.

• Cross-linking Mass Spectrometry: This approach can identify interaction interfaces with binding partners, providing insights into functional mechanisms.