Recombinant Microcystis aeruginosa UPF0754 membrane protein MAE_37850 (MAE_37850)

What is the MAE_37850 protein and what are its basic structural characteristics?

MAE_37850 is a UPF0754 family membrane protein from the cyanobacterium Microcystis aeruginosa (strain NIES-843). It is characterized as a multi-pass membrane protein located in the cell inner membrane . The protein consists of 412 amino acids in its full-length form and contains multiple transmembrane regions that span the lipid bilayer . According to UniProt database (entry B0JPN3), MAE_37850 belongs to the UPF0754 protein family, which remains functionally uncharacterized despite structural data being available . The amino acid sequence includes hydrophobic regions consistent with membrane integration, and the protein exhibits structural motifs common to transmembrane proteins with multiple spanning regions.

How is recombinant MAE_37850 typically produced for research purposes?

Recombinant MAE_37850 is primarily produced using E. coli expression systems . The protein is typically expressed with an N-terminal tag (often a 10xHis-tag) to facilitate purification . The expression construct includes the complete or partial coding sequence of MAE_37850 cloned into an appropriate expression vector. Following expression in E. coli, the protein is extracted from bacterial membranes or inclusion bodies using detergents or denaturing agents, purified via affinity chromatography (exploiting the His-tag), and subsequently refolded if necessary. The purified protein achieves >85% purity as confirmed by SDS-PAGE analysis . For research applications, the protein is commonly supplied in either liquid form in Tris/PBS-based buffer (pH 8.0) with 6% trehalose or as a lyophilized powder for reconstitution .

What storage and handling protocols ensure optimal stability of recombinant MAE_37850?

Optimal storage conditions for recombinant MAE_37850 vary depending on the preparation format. For long-term storage, the protein should be kept at -20°C or preferably -80°C . The liquid form has a typical shelf life of 6 months, while the lyophilized powder remains stable for approximately 12 months when properly stored at these temperatures . To maintain protein integrity, it is advisable to add glycerol (5-50% final concentration, with 50% being recommended) before aliquoting and freezing .

For working with the protein, researchers should avoid repeated freeze-thaw cycles as these can significantly compromise protein structure and function . Working aliquots can be stored at 4°C for up to one week . When reconstituting lyophilized protein, it is recommended to briefly centrifuge the vial before opening to collect all material at the bottom. Reconstitution should be performed using deionized sterile water to achieve a final concentration of 0.1-1.0 mg/mL . After reconstitution, the solution should be gently mixed until completely solubilized, avoiding vigorous shaking or vortexing that might denature the membrane protein.

What experimental approaches are used to confirm the membrane localization of MAE_37850?

Confirming the membrane localization of MAE_37850 typically involves multiple complementary approaches. Computational prediction tools provide initial evidence of transmembrane domains based on hydrophobicity analysis of the amino acid sequence . Experimentally, subcellular fractionation techniques are employed to isolate membrane fractions from E. coli expressing the recombinant protein, followed by Western blotting using anti-His antibodies to detect the tagged protein in membrane preparations.

Fluorescence microscopy using GFP or other fluorescent protein fusions can visualize the localization pattern in both bacterial and mammalian expression systems. This approach is supported by observations from similar transmembrane protein studies showing that designed membrane proteins successfully localize to the plasma membrane in both bacterial and mammalian cells . Additional confirmation may come from protease protection assays, where differential susceptibility to proteolytic digestion indicates membrane-embedded regions. Circular dichroism spectroscopy can provide evidence of the characteristic α-helical secondary structure common in transmembrane proteins. For definitive structural characterization, techniques similar to those used for other transmembrane proteins, such as X-ray crystallography or cryo-electron microscopy, would be required to resolve the protein's membrane topology and confirm its multi-pass nature .

How does the structure of MAE_37850 compare to other computationally designed membrane proteins?

While the native MAE_37850 is not a computationally designed protein, its structural characteristics can be compared to recent advances in de novo designed membrane proteins. MAE_37850 contains multiple membrane-spanning regions as a natural multi-pass membrane protein . Recent breakthrough research from the Baker lab demonstrates that computationally designed transmembrane proteins with multiple membrane-spanning regions can now be created with high accuracy . These designed proteins form stable structures with two to four membrane-spanning regions per subunit, similar to the topology predicted for MAE_37850 .

The computational design principles applied to synthetic membrane proteins focus on creating stable hydrophobic interactions within the membrane environment and favorable electrostatic interactions in the aqueous regions . Crystal structures of these designed proteins reveal "rocket-shaped" architectures with wide cytoplasmic bases that funnel into transmembrane helical bundles . It would be informative to determine whether MAE_37850 adopts similar structural motifs. These designed proteins demonstrate excellent stability in membrane environments, as confirmed through magnetic tweezer unfolding experiments . Comparative stability studies between natural membrane proteins like MAE_37850 and de novo designed proteins could provide valuable insights into evolutionary versus rational design principles for membrane protein architecture.

What is the potential functional relationship between MAE_37850 and other proteins in Microcystis aeruginosa?

The functional role of MAE_37850 remains largely uncharacterized, but insights may be gained by examining its genomic context and potential interactions with other proteins in Microcystis aeruginosa. As a member of the UPF0754 family, it likely participates in processes specific to cyanobacteria . Comparative genomic analyses using tools such as STRING database (referenced as STRING:449447.MAE_37850) may reveal co-expression patterns or genomic proximity to genes with known functions .

Microcystis aeruginosa produces numerous bioactive compounds, including the hepatotoxic microcystin and a diverse array of metabolites with various biological activities . Though MAE_37850 is not directly implicated in microcystin production, membrane proteins often participate in transport, sensing, or secretion pathways relevant to secondary metabolite production. Interestingly, research on Microcystis aeruginosa has identified phycobiliprotein complexes as significant immunogenic components that can induce allergic sensitization in susceptible individuals . These immunogenic properties appear to be modulated by microcystin in a dose-dependent manner . Given MAE_37850's membrane localization, it could potentially contribute to cell surface properties that influence the organism's interaction with host immune systems or environmental stimuli. Comprehensive protein-protein interaction studies, perhaps using affinity purification coupled with mass spectrometry, would be necessary to elucidate MAE_37850's position within the functional protein network of Microcystis aeruginosa.

What experimental approaches would be most effective for determining the function of MAE_37850?

Determining the function of uncharacterized membrane proteins like MAE_37850 requires a multi-faceted approach. Beginning with bioinformatic analyses, researchers should perform thorough sequence homology searches, domain predictions, and structural modeling to identify potential functional motifs or similarities to characterized proteins. Tools like KEGG pathway analysis (referenced as KEGG:mar:MAE_37850) can suggest metabolic or signaling pathways where this protein might function .

Experimentally, gene knockout or CRISPR-mediated gene editing in Microcystis aeruginosa followed by phenotypic characterization could reveal the physiological impact of MAE_37850 loss. Complementary to this, heterologous overexpression systems could be used to assess effects on membrane permeability, cell morphology, or response to environmental stressors. Protein-protein interaction studies using techniques like bacterial two-hybrid systems, co-immunoprecipitation, or proximity labeling methods would identify interaction partners that might suggest functional associations.

For biochemical characterization, purified recombinant MAE_37850 could be reconstituted into liposomes or nanodiscs to assess potential transport activity, channel formation, or enzymatic functions. Isothermal titration calorimetry or surface plasmon resonance could identify small molecule binding partners. Structural studies using X-ray crystallography, cryo-electron microscopy, or nuclear magnetic resonance spectroscopy would provide atomic-level insights into functional domains. Additionally, comparing gene expression patterns across different growth conditions or stress responses might reveal conditions where MAE_37850 is particularly important, guiding more focused functional investigations.

How might recombinant MAE_37850 be utilized in immunological research related to cyanobacterial exposure?

Recombinant MAE_37850 represents a valuable tool for investigating immune responses to cyanobacterial proteins. Research has demonstrated that specific IgE responses can be elicited by non-toxic strains of Microcystis aeruginosa in patients with chronic rhinitis, indicating that allergenicity resides in non-toxin components of the organism . While phycobiliprotein complexes have been identified as primary sensitizing agents, membrane proteins like MAE_37850 may also contribute to immunogenicity.

To investigate this possibility, researchers could develop ELISA-based assays using purified recombinant MAE_37850 to screen serum samples from individuals exposed to cyanobacteria for specific IgE, IgG, or other immunoglobulin responses . Such assays would help determine whether MAE_37850 contributes to the allergenic profile of Microcystis aeruginosa. For mechanistic studies, recombinant MAE_37850 could be used in cell-based assays with rat basophil leukemia cells (similar to those used for phycobiliproteins) to assess its capacity to induce degranulation and mediator release .

The interesting finding that microcystin toxin inhibits the allergenicity of certain Microcystis aeruginosa proteins in a dose-dependent manner suggests complex interactions between toxins and immunogenic proteins . Similar inhibition studies could be performed with MAE_37850 to determine whether its potential immunogenicity is likewise modulated by microcystin. Additionally, epitope mapping using overlapping peptide arrays derived from MAE_37850 could identify specific regions of the protein responsible for any observed immunogenicity, informing both basic immunology research and potential development of diagnostic tools for cyanobacterial exposure.

What purification strategies are most effective for obtaining high-quality recombinant MAE_37850?

Purifying membrane proteins like MAE_37850 presents significant challenges due to their hydrophobic nature and tendency to aggregate outside their native membrane environment. The most effective purification strategy begins with optimizing the expression system. While E. coli is commonly used , specific strains designed for membrane protein expression (such as C41/C43 or Lemo21) may yield better results. Induction conditions should be carefully optimized, often using lower temperatures (16-25°C) and inducer concentrations to promote proper folding.

For extraction, the choice of detergent is critical. A screen of mild non-ionic detergents (DDM, LMNG, or digitonin) and zwitterionic detergents (CHAPS, Fos-choline) should be performed to identify optimal solubilization conditions that maintain native protein structure. The presence of an N-terminal His-tag facilitates initial purification via immobilized metal affinity chromatography (IMAC) . To achieve >85% purity as reported in product specifications , additional purification steps are typically required, which may include size exclusion chromatography to remove aggregates and ion exchange chromatography for further enrichment.

For structural or functional studies requiring especially high purity, techniques such as affinity purification with a secondary tag (such as FLAG or Strep-tag II) may be employed following IMAC. Quality control assessments should include SDS-PAGE, Western blotting, and analytical size exclusion chromatography to confirm purity, identity, and monodispersity. For functional studies, reconstitution into proteoliposomes or lipid nanodiscs provides a more native-like membrane environment than detergent micelles. Throughout the purification process, stability should be monitored using techniques such as differential scanning fluorimetry to ensure the protein remains properly folded.

What considerations are important when designing experiments to study protein-protein interactions involving MAE_37850?

Studying protein-protein interactions involving membrane proteins like MAE_37850 requires specialized approaches that preserve the native conformational state of the protein. When designing such experiments, researchers should first consider the membrane environment. Traditional yeast two-hybrid systems are poorly suited for membrane proteins; instead, specialized membrane yeast two-hybrid, bacterial two-hybrid, or split-ubiquitin systems should be employed for initial screening of potential interaction partners.

For biochemical validation of interactions, co-immunoprecipitation experiments should be performed under conditions that maintain membrane protein solubility, typically using crosslinking agents followed by detergent solubilization. The choice of detergent is crucial—mild options like digitonin or LMNG that preserve protein-protein interactions should be prioritized. Alternatively, proximity-based labeling methods such as BioID or APEX2 can identify proteins in close proximity to MAE_37850 in living cells without requiring solubilization.

When using recombinant MAE_37850, reconstitution into membrane mimetics such as nanodiscs provides a more native-like environment for interaction studies compared to detergent solutions. Surface plasmon resonance or microscale thermophoresis can then assess binding kinetics with potential partners. For structural characterization of complexes, techniques such as cryo-electron microscopy are particularly valuable for membrane protein complexes that may be challenging to crystallize.

Researchers should also consider the potential effects of the His-tag on interactions; control experiments with tag-cleaved protein or alternatively tagged constructs can address this concern . Finally, bioinformatic approaches using tools like STRING database analysis (referenced as STRING:449447.MAE_37850) can provide theoretical interaction networks to guide experimental design . Validation of interactions in the native organism (Microcystis aeruginosa) is ultimately important to confirm biological relevance of any interactions identified in heterologous systems.

How can researchers optimize expression systems for functional studies of MAE_37850?

Optimizing expression systems for functional studies of membrane proteins like MAE_37850 requires careful consideration of multiple factors to ensure proper folding and functionality. While E. coli is commonly used for initial expression , researchers conducting functional studies should evaluate alternative expression hosts. For prokaryotic expression, specialized E. coli strains like C41/C43(DE3), which are adapted for membrane protein expression, may yield better results than standard BL21 derivatives. For eukaryotic membranes, yeast systems (Pichia pastoris or Saccharomyces cerevisiae), insect cells (via baculovirus), or mammalian cell lines might provide more appropriate membrane environments.

Expression construct design significantly impacts success. While the N-terminal His-tag used in commercial preparations facilitates purification , its position or presence may affect function. Researchers should consider creating constructs with cleavable tags or C-terminal tags as alternatives. The expression vector should allow tight control over expression levels, as membrane protein overexpression often leads to toxicity and aggregation. Inducible promoters with variable strength can help fine-tune expression levels.

Induction conditions require optimization: lower temperatures (16-18°C), reduced inducer concentrations, and extended expression times often improve folding of membrane proteins. Supplementing growth media with specific lipids or membrane components may enhance proper insertion into membranes. For functional assays in particular, preserving the native membrane environment is critical. Techniques such as fluorescent reporter systems to monitor localization, proteoliposome reconstitution for transport or channel studies, or cell-based activity assays can provide more relevant functional data than studies with detergent-solubilized protein.

When expressing MAE_37850 in heterologous systems, codon optimization for the host organism may improve translation efficiency. Additionally, co-expression with appropriate chaperones can enhance folding and membrane insertion. For mammalian expression, the finding that designed multi-pass membrane proteins successfully localize to the plasma membrane in mammalian cells suggests that MAE_37850 could potentially be expressed in these systems for functional studies .

How could MAE_37850 contribute to understanding membrane protein evolution in cyanobacteria?

MAE_37850 represents an interesting subject for evolutionary studies of membrane proteins in cyanobacteria. As a member of the UPF0754 protein family , which remains functionally uncharacterized despite having structural information available, MAE_37850 could provide insights into how membrane protein families evolve specialized functions in photosynthetic organisms. Comparative genomic analyses across diverse cyanobacterial species would reveal the conservation patterns of UPF0754 family proteins, potentially identifying core functional regions through patterns of evolutionary constraint.

The multi-pass membrane topology of MAE_37850 makes it an interesting counterpoint to the recent advances in de novo designed membrane proteins . Comparing the structural features of naturally evolved membrane proteins like MAE_37850 with computationally designed proteins could reveal convergent solutions to the biophysical challenges of membrane integration. This comparison might highlight how evolutionary processes and rational design principles arrive at similar or different architectural solutions for stable membrane protein folding.

Microcystis aeruginosa has ecological importance as a bloom-forming cyanobacterium with significant environmental impact . Understanding the evolution of its membrane proteome, including proteins like MAE_37850, could provide insights into the adaptive mechanisms that enable its successful colonization of diverse aquatic environments. Additionally, investigating potential horizontal gene transfer events involving MAE_37850 homologs might reveal how membrane protein innovations spread across microbial communities in aquatic ecosystems.

Future research could employ ancestral sequence reconstruction and resurrection approaches to experimentally characterize the functional properties of inferred ancestral forms of MAE_37850, potentially revealing how its structure and function have evolved over time. This evolutionary perspective would complement the growing field of de novo membrane protein design , potentially inspiring new approaches for designing membrane proteins with specific functional properties.

What role might MAE_37850 play in the environmental adaptation of Microcystis aeruginosa?

As a membrane protein in Microcystis aeruginosa, MAE_37850 potentially contributes to the organism's ability to sense and respond to environmental conditions, though its specific function remains uncharacterized . Membrane proteins often serve as environmental sensors, transporters, or components of signaling pathways that enable adaptation to changing conditions. Given that Microcystis aeruginosa is known for forming harmful algal blooms under specific environmental conditions, membrane proteins like MAE_37850 may play roles in the cellular responses that trigger bloom formation.

Research approaches to investigate this potential role could include comparative expression studies of MAE_37850 under various environmental conditions (varying temperature, light intensity, nutrient availability, or salinity) using quantitative PCR or proteomics. Gene knockout or silencing studies followed by phenotypic characterization under stress conditions could reveal whether MAE_37850 contributes to stress tolerance or environmental sensing. As Microcystis aeruginosa produces various bioactive compounds including microcystin in response to environmental cues , investigating whether MAE_37850 influences toxin production pathways would be particularly relevant.

The observed interplay between toxicity and immunogenicity in Microcystis aeruginosa suggests complex regulatory networks governing how these organisms interact with their environment. If MAE_37850 participates in environmental sensing or response pathways, it might indirectly influence these interactions. Climate change is altering aquatic ecosystems in ways that may favor cyanobacterial blooms, making it increasingly important to understand the molecular mechanisms, potentially including MAE_37850, that contribute to the environmental adaptation and success of bloom-forming species like Microcystis aeruginosa.

How can structural studies of MAE_37850 contribute to membrane protein design principles?

Structural studies of naturally occurring membrane proteins like MAE_37850 provide valuable reference points for improving computational design approaches for membrane proteins. While significant advances have been made in designing multi-pass membrane proteins , naturally evolved proteins offer examples of solutions to the challenges of membrane integration that have been refined through evolutionary selection. Determining the high-resolution structure of MAE_37850 using X-ray crystallography, cryo-electron microscopy, or nuclear magnetic resonance spectroscopy would provide insights into how this protein achieves stable membrane integration with multiple spanning regions.

Comparison between the structural features of MAE_37850 and successfully designed membrane proteins could reveal common principles and important differences in how transmembrane helices pack together, how loops between helices are structured, and how hydrophobic mismatch at the membrane-water interface is resolved. The "rocket-shaped structure with a wide cytoplasmic base that funnels into transmembrane helices" described for designed membrane proteins represents one successful architectural solution; analyzing whether natural proteins like MAE_37850 adopt similar or different architectures would be informative.

Stability studies of MAE_37850 using techniques like differential scanning calorimetry or hydrogen-deuterium exchange mass spectrometry could reveal how natural membrane proteins achieve thermodynamic stability in the membrane environment. These insights could inform the energy functions used in computational protein design. Additionally, investigating how MAE_37850 folds and inserts into membranes could provide insights for designing proteins that efficiently integrate into cellular membranes in expression systems. The reported success of designed membrane proteins localizing to plasma membranes in both bacterial and mammalian cells suggests shared principles of membrane integration that may be further refined through studies of natural membrane proteins like MAE_37850.