Recombinant Mycobacterium marinum UPF0233 membrane protein MMAR_0013 (MMAR_0013)

Intermediate Research Questions

• What expression systems are most effective for producing recombinant MMAR_0013?

The expression of membrane proteins like MMAR_0013 presents significant challenges due to their hydrophobic nature and requirements for proper membrane insertion. Based on empirical research, several expression systems have been evaluated:

For MMAR_0013 specifically, E. coli expression systems using the BL21 strain have been documented with His-tagging for purification purposes . The experimental design approach using factorial designs has been particularly effective, allowing systematic optimization of culture conditions with fewer experiments .

Recent advances in vesicle-packaged recombinant protein production show promise for membrane proteins like MMAR_0013, as they enable production of proteins in a membrane environment that mimics their native state, potentially improving folding and functionality .

• How do you optimize solubility when expressing recombinant MMAR_0013?

Optimizing solubility for membrane proteins requires a multifactorial approach targeting several key parameters simultaneously. For MMAR_0013, the following optimization strategy is recommended:

Statistical experimental design has proven particularly valuable for membrane protein expression, allowing researchers to evaluate multiple variables simultaneously and identify significant interactions between parameters that affect solubility . Studies have shown that using fractional factorial designs can increase soluble protein yields by 2-5 fold compared to univariate optimization approaches.

Additionally, computational design approaches have recently demonstrated success in creating soluble analogues of integral membrane proteins that maintain their structural features in solution , which could be an alternative approach for MMAR_0013 if direct expression proves challenging.

• What purification methods work best for recombinant MMAR_0013?

Purification of membrane proteins like MMAR_0013 requires specialized approaches to maintain protein stability and functionality. A recommended purification workflow includes:

• Cell lysis optimization: Use of specialized detergents (n-dodecyl-β-D-maltoside or CHAPS at 1-2%) that effectively solubilize membrane proteins while preserving native structure.

• Affinity chromatography: Utilizing the His-tag engineered onto recombinant MMAR_0013 for immobilized metal affinity chromatography (IMAC) . Optimal conditions include:

  • Nickel or cobalt resin

  • Binding buffer containing 20 mM imidazole to reduce non-specific binding

  • Washing with 40-60 mM imidazole

  • Elution with 250-500 mM imidazole

• Size exclusion chromatography: Secondary purification step to separate protein based on size, ensuring removal of aggregates and contaminants.

• Quality control assessment: SDS-PAGE analysis showing >90% purity is typically achieved through this workflow .

For vesicle-packaged expression systems, the purification process is simplified as the protein remains in a membrane environment throughout, potentially improving stability . This approach allows isolation of protein-filled vesicles through filtration or centrifugation steps, with yields of up to 2.5 g/L of bacterial culture reported for membrane proteins .

• What analytical methods are suitable for characterizing recombinant MMAR_0013?

Comprehensive characterization of MMAR_0013 requires multiple analytical approaches targeting different protein properties:

For functional characterization, incorporation into liposomes or nanodiscs followed by biophysical techniques or binding assays would be appropriate. Recent multiplexed protein analysis platforms such as Olink and Luminex can also be employed for specific interaction studies , though optimization for membrane proteins is required.

Advanced Research Questions

• How does MMAR_0013 potentially contribute to Mycobacterium marinum pathogenesis?

While direct evidence for MMAR_0013's role in pathogenesis is limited in current literature, several research avenues can be explored based on understanding of mycobacterial pathogenesis and membrane proteins:

M. marinum serves as a well-established model for studying mycobacterial pathogenesis, sharing many virulence mechanisms with M. tuberculosis . Membrane proteins often play critical roles in bacterial adaptation to host environments, immune evasion, and virulence factor secretion.

Research approaches to investigate MMAR_0013's potential role in pathogenesis include:

• Comparative infection studies: Comparing wild-type and MMAR_0013 deletion mutants in cellular and animal models to assess effects on bacterial survival, replication, and virulence. The zebrafish model provides an excellent system for such studies, allowing visualization of granuloma formation and bacterial dissemination .

• Interaction with host components: Investigating whether MMAR_0013 interacts with host cell receptors or immune components using co-immunoprecipitation and surface plasmon resonance.

• Membrane integrity assessment: Determining whether MMAR_0013 affects mycobacterial membrane permeability or resistance to host defense mechanisms like antimicrobial peptides.

• Association with secretion systems: Examining potential connections between MMAR_0013 and the ESX-1 secretion system, which is crucial for M. marinum virulence and phagosomal escape .

• What genetic manipulation techniques work best for studying MMAR_0013 function?

Genetic manipulation of mycobacterial genes requires specialized techniques due to their complex cell walls and relatively slow growth. For MMAR_0013, several approaches have been validated:

• Creation of unmarked deletion mutants: Using homologous recombination to generate clean gene deletions without antibiotic resistance markers, allowing precise assessment of gene function without polar effects on neighboring genes .

• Complementation strategies: Reintroducing the wild-type gene or specific mutants on integrative or replicative plasmids to confirm phenotype specificity and perform structure-function analyses .

• Site-directed mutagenesis: Targeting specific amino acids to identify critical residues for protein function, particularly within predicted functional domains or membrane-spanning regions.

• Reporter fusions: Creating translational fusions with fluorescent proteins to study localization patterns and expression levels under different conditions.

• Controlled expression systems: Using inducible promoters to modulate expression levels and study dose-dependent effects.

Importantly, whole-genome sequencing should be performed on generated mutants to identify any secondary mutations that might affect phenotype interpretation, as demonstrated in research on other M. marinum genes .

• How can experimental approaches reveal MMAR_0013's role in mycobacterial membrane biology?

Investigating MMAR_0013's role in membrane biology requires specialized techniques that can probe protein-membrane interactions and functional consequences:

• Membrane microdomain analysis: Recent research has shown that membrane microdomains are crucial for M. marinum pathogenesis . Techniques like detergent-resistant membrane isolation, sterol depletion, and disruption of microdomain organizing proteins could reveal whether MMAR_0013 localizes to or influences these structures.

• Lipid interaction studies: Using fluorescence anisotropy or surface plasmon resonance to determine specific lipid binding preferences of purified MMAR_0013.

• Membrane permeability assessment: Comparing wild-type and MMAR_0013 mutant strains for differences in membrane permeability to various compounds (antibiotics, dyes), which could indicate altered membrane organization.

• Super-resolution microscopy: Techniques like PALM or STORM combined with fluorescently tagged MMAR_0013 can reveal nanoscale distribution patterns within the membrane.

• Cryo-electron tomography: Visualizing membrane architecture in wild-type versus mutant strains to identify structural alterations.

Such approaches could determine whether MMAR_0013 plays a structural role in membrane organization, contributes to membrane domain formation, or affects the distribution of other membrane components critical for mycobacterial physiology and pathogenesis.

• How might MMAR_0013 interact with other proteins in the mycobacterial membrane?

Identifying protein-protein interactions involving MMAR_0013 would provide critical insights into its functional networks. Several complementary approaches can be employed:

• Pull-down assays: Using His-tagged recombinant MMAR_0013 as bait to identify binding partners from mycobacterial lysates, followed by mass spectrometry identification.

• Bacterial two-hybrid systems: Modified for membrane proteins to screen for potential interaction partners.

• Co-immunoprecipitation: Using antibodies against MMAR_0013 or epitope-tagged versions to isolate protein complexes from mycobacterial lysates.

• Proximity labeling techniques: Using BioID or APEX2 fusions to identify proteins in close proximity to MMAR_0013 in living bacteria.

• Crosslinking mass spectrometry: Employing chemical crosslinkers to capture transient interactions followed by MS identification.

Based on its predicted role in cell division (annotated as CrgA), MMAR_0013 might interact with components of the mycobacterial divisome. It might also associate with membrane remodeling proteins or components of cell wall biosynthesis pathways. Comparative interactomics between wild-type and mutated versions could highlight interaction domains critical for function.

• How can structural biology approaches advance our understanding of MMAR_0013?

Determining the three-dimensional structure of membrane proteins presents significant challenges, but recent advances offer promising approaches for MMAR_0013:

• Computational structural prediction: Recent deep learning approaches like AlphaFold2 have dramatically improved membrane protein structure prediction accuracy. These computational models can provide initial structural hypotheses for MMAR_0013.

• Creation of soluble analogues: As demonstrated for other membrane proteins, computational design approaches can create soluble versions that retain key structural features while facilitating crystallization . These "solubilized" versions maintain native folds but with reduced hydrophobicity.

• Cryo-electron microscopy: For purified MMAR_0013 in membrane mimetics like nanodiscs or detergent micelles, single-particle cryo-EM can potentially determine structure without crystallization.

• Solid-state NMR: For membrane proteins reconstituted in lipid bilayers, solid-state NMR can provide structural constraints and dynamics information.

• EPR spectroscopy: Site-directed spin labeling combined with EPR can reveal topological information and conformational changes.

The structural data would significantly advance understanding of how MMAR_0013 functions at the molecular level, potentially revealing binding sites for small molecules that could modulate its activity, which would be valuable if it proves to be involved in pathogenesis.

• What are the challenges in working with recombinant MMAR_0013 and how can they be overcome?

Membrane proteins like MMAR_0013 present several technical challenges that require specialized approaches:

The innovative vesicle-packaged recombinant protein production system described in search result offers a promising approach for MMAR_0013, as it allows the protein to remain in a membrane environment throughout expression and purification. This system has demonstrated success with challenging membrane proteins, yielding up to 2.5 g/L of functional protein .

• How can single-molecule techniques reveal functional aspects of MMAR_0013?

Single-molecule approaches offer unique insights into membrane protein dynamics and interactions not accessible through bulk measurements:

• Single-molecule FRET: By labeling specific residues on MMAR_0013 with fluorophore pairs, conformational changes and dynamics can be monitored in real-time, revealing potential functional states.

• Single-molecule force spectroscopy: Using atomic force microscopy (AFM) to measure unfolding forces and energy landscapes, providing insights into structural stability and domains.

• Single-molecule tracking: Following fluorescently labeled MMAR_0013 in live bacteria to determine diffusion characteristics and potential confinement to specific membrane domains.

• Single-molecule proteomics: As highlighted in recent advances , detecting and analyzing individual protein molecules can identify rare post-translational modifications or conformational states that might be critical for function.

• Nanopore analysis: If MMAR_0013 forms pores or channels, single-channel recordings can characterize conductance properties and substrate specificity.

These approaches would be particularly valuable for understanding dynamic aspects of MMAR_0013 function that might be averaged out in ensemble measurements, potentially revealing heterogeneity in behavior that relates to different functional states within the bacterial membrane.

Importance of MMAR_0013 in Mycobacterial Research

While the specific function of MMAR_0013 requires further characterization, its study offers several important advantages. As a membrane protein in Mycobacterium marinum, it provides insights into membrane biology relevant to pathogenic mycobacteria. The protein's relatively small size (93 amino acids) makes it tractable for structural studies while still representing the challenges typical of membrane proteins.