Recombinant Uncharacterized membrane protein Rv3691 (Rv3691)

What is Rv3691 and what organism does it come from?

Rv3691 is an uncharacterized membrane protein encoded by the Rv3691 gene in the genome of Mycobacterium tuberculosis, the causative agent of tuberculosis . The protein is specifically found in the reference strain ATCC 25618 / H37Rv, which is widely used in tuberculosis research . It is classified as a membrane protein with 381 amino acids in its full-length form and has been assigned the UniProt identifier O69659 .

The complete amino acid sequence of Rv3691 is: MGAGPVIPTRLATVRRRRPWRGVLLTLAAVAVVASIGTYLTAPRPGGAMAPASTSSTGGHALATLLGNHGVEVVVADSIADVEAAARPDSLLLVAQTQYLVDNALLDRLAKAPGDLLLVAPTSRTRTALTPQLRIAAASPFNSQPNCTLREANRAGSVQWGPSDTYQATGDLVLTSCYGGALVRFRAEGRTITVVGSSNFMTNGGLLPAGNAALAMNLAGNRPRLVWYAPDHIEGEMSSPSSLSDLIPENVHWTIWQLWLVVLLVALWKGRRIGPLVAEELPVVIRASETVEGRGRLYRSRRARDRAADALRTATLQRLRPRLGVGAGAPAPAVVTTIAQRSKADPPFVAYHLFGPAPATDNDLLQLARALDDIERQVTHS . Understanding this sequence is essential for designing experiments to investigate the protein's structure and function.

What expression systems are used to produce recombinant Rv3691?

Multiple expression systems have been developed for producing recombinant Rv3691 protein, each with distinct advantages depending on the research application. The primary expression systems documented in the literature include Escherichia coli and yeast-based expression systems .

For E. coli-based expression, the full-length Rv3691 (amino acids 1-381) is typically fused to an N-terminal His-tag to facilitate purification . This approach is advantageous for producing large quantities of protein for structural and biochemical studies. The E. coli system is particularly suitable when the research requires complete protein coverage for applications such as antibody production or full structural analysis.

Alternatively, yeast-based expression systems have been employed to produce partial constructs of Rv3691 . This approach may be beneficial for expressing specific domains or when post-translational modifications more similar to those in eukaryotic systems are desired. When working with membrane proteins like Rv3691, yeast systems sometimes provide better folding environments that more closely resemble eukaryotic membranes, potentially resulting in more native-like protein conformations.

What are the optimal storage conditions for recombinant Rv3691?

Proper storage of recombinant Rv3691 is critical for maintaining protein stability and functionality. The protein is typically supplied either as a lyophilized powder or in liquid form with specific buffer components . For long-term storage, the following conditions are recommended:

Lyophilized powder forms of Rv3691 maintain stability for approximately 12 months when stored at -20°C or -80°C . This format offers extended shelf life compared to liquid preparations and is preferred for long-term storage.

For liquid formulations, the recommended storage period is typically 6 months at -20°C or -80°C . The protein is often provided in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 to enhance stability .

To prevent protein degradation from repeated freeze-thaw cycles, it is strongly recommended to prepare small working aliquots upon initial reconstitution . These working aliquots can be maintained at 4°C for up to one week for ongoing experiments . For reconstitution, it is advisable to briefly centrifuge the vial before opening to collect all material at the bottom, then dissolve the protein in deionized sterile water to a concentration between 0.1-1.0 mg/mL . Adding glycerol to a final concentration of 5-50% is recommended before aliquoting for long-term storage, with 50% being a standard reference concentration .

What is known about genetic variations in Rv3691 and their potential functional impact?

Genetic diversity studies of Mycobacterium tuberculosis have identified several mutations in the Rv3691 gene across different clinical and laboratory strains. One notable polymorphism is the A62G amino acid substitution that has been documented in laboratory stocks of the H37Rv strain . This substitution results in the replacement of alanine with glycine at position 62 in the protein sequence.

The A62G mutation has been computationally predicted to have a "Neutral" impact on protein function, suggesting that this particular substitution might not significantly alter the protein's core functionality . This classification was likely determined using predictive algorithms that assess the biochemical properties of the substituted amino acids and their location within the protein structure. Despite being classified as neutral, such mutations can still potentially influence protein-protein interactions, membrane localization, or subtle functional aspects not captured by predictive algorithms.

In broader studies examining M. tuberculosis genomic evolution, researchers have found that T cell immunity may constrain genetic diversity during infection . When comparing bacterial populations passaged in T cell-deficient versus immunocompetent mice, the presence of T cells was associated with increased diversity in the M. tuberculosis genome . This suggests that immune pressure might influence the evolution of membrane proteins like Rv3691, potentially selecting for variants that can evade immune recognition while maintaining essential functions.

What experimental approaches can be used to characterize uncharacterized membrane proteins like Rv3691?

Characterizing uncharacterized membrane proteins such as Rv3691 requires a multi-faceted approach that combines structural, functional, and computational methods. A systematic experimental workflow would typically include:

Structural Characterization Methodologies

For membrane proteins like Rv3691, structural determination presents significant challenges due to their hydrophobic nature and requirement for lipid environments. X-ray crystallography remains challenging but feasible with appropriate detergent screening and crystallization conditions. Cryo-electron microscopy (cryo-EM) has emerged as a powerful alternative that doesn't require crystallization and can visualize the protein in a more native-like environment.

Nuclear Magnetic Resonance (NMR) spectroscopy can provide valuable information about protein dynamics and ligand interactions, particularly for specific domains or regions of interest. For initial topology assessment, techniques such as limited proteolysis combined with mass spectrometry can help identify exposed regions versus membrane-embedded domains of Rv3691.

Functional Analysis Approaches

Since Rv3691 is uncharacterized, functional studies should begin with comparative analyses against proteins of known function. Phenotypic assays using knockout strains (ΔRv3691) can reveal the protein's role in bacterial growth, survival under stress conditions, or virulence. Complementation studies with wild-type and mutated versions (including the documented A62G variant) would confirm functional impacts of specific residues.

Protein-protein interaction studies using techniques such as bacterial two-hybrid systems, co-immunoprecipitation, or proximity labeling methods can identify binding partners and potentially place Rv3691 within known cellular pathways. Subcellular localization studies using fluorescently tagged versions can confirm membrane association and specific distribution patterns within the bacterial cell.

How might Rv3691 relate to M. tuberculosis pathogenesis and virulence?

While direct evidence linking Rv3691 to M. tuberculosis pathogenesis is limited in the available literature, several lines of investigation could establish such connections. As a membrane protein, Rv3691 could potentially function in host-pathogen interactions, nutrient acquisition, or stress response pathways that contribute to bacterial survival during infection.

Studies examining M. tuberculosis genomic diversity have shown that bacterial populations accumulate mutations during in vitro culture, particularly in genes that are not essential for growth in laboratory media but might be important for in vivo survival . The presence of Rv3691 variants in clinical isolates and laboratory strains suggests it may be subject to selective pressures during infection or transmission.

The genetic diversity observed in M. tuberculosis during infection in immunocompetent versus T cell-deficient mice indicates that T cell immunity shapes bacterial evolution . If Rv3691 is involved in interactions with the host immune system, it might be under selective pressure to evade recognition while maintaining essential functions. This hypothesis could be tested through epitope mapping and analysis of Rv3691 sequence conservation across clinical isolates.

What purification strategies are optimal for recombinant membrane proteins like Rv3691?

Purifying membrane proteins presents unique challenges compared to soluble proteins due to their hydrophobic nature and requirement for detergents or lipid environments. For Rv3691, several purification strategies can be employed, with the optimal approach depending on the specific research application.

Affinity Chromatography Optimization

The His-tagged recombinant Rv3691 expressed in E. coli can be purified using immobilized metal affinity chromatography (IMAC) . This approach typically uses Ni-NTA or Co-NTA resins to capture the His-tagged protein. Critical factors for optimizing IMAC purification include:

• Detergent selection: Screening multiple detergents (e.g., n-dodecyl-β-D-maltoside, digitonin, CHAPS) at concentrations above their critical micelle concentration is essential for efficient extraction while maintaining protein stability.

• Buffer composition: Including glycerol (10-20%) and stabilizing agents like trehalose (as used in commercial preparations at 6%) can significantly enhance protein stability during purification.

• Elution conditions: Using either an imidazole gradient or pH step gradient for elution, with careful optimization to minimize co-purification of contaminants.

Secondary Purification Steps

Following initial affinity purification, additional chromatography steps can enhance purity beyond the 90% level typically achieved with single-step purification . Size exclusion chromatography is particularly valuable for membrane proteins as it can separate monomeric protein from aggregates and remove empty detergent micelles. Ion exchange chromatography may provide further purification based on the protein's charge properties at specific pH values.

How can researchers design experiments to investigate potential functions of Rv3691?

Given the uncharacterized nature of Rv3691, a systematic approach to functional investigation would involve both computational predictions and experimental validation. The following experimental design framework provides a comprehensive strategy:

Computational Function Prediction

Begin with bioinformatic analyses to generate functional hypotheses. Sequence-based approaches should include:

• Homology detection using PSI-BLAST, HHpred, and AlphaFold to identify distant relatives with known functions.

• Transmembrane topology prediction using tools like TMHMM, Phobius, and TOPCONS to map membrane-spanning regions.

• Domain and motif scanning to identify functional elements using InterProScan and MOTIF Search.

• Evolutionary analysis to identify conserved residues that may be functionally important, particularly focusing on the region containing the documented A62G substitution .

Experimental Validation Strategy

Based on computational predictions, design targeted experiments to test specific functional hypotheses:

• Gene deletion and complementation studies to assess the effect of Rv3691 on bacterial growth, stress response, and virulence in animal models.

• Site-directed mutagenesis focusing on highly conserved residues to identify those critical for function.

• Conditional expression systems to study the effects of Rv3691 depletion or overexpression on cellular processes and morphology.

• Protein localization studies using fluorescent protein fusions or immunolocalization to determine subcellular distribution and potential changes during different growth phases or stress conditions.

What techniques are appropriate for studying protein-protein interactions involving Rv3691?

Understanding the interactome of Rv3691 could provide valuable insights into its cellular function. Several complementary approaches can be employed to identify and validate protein interaction partners:

Unbiased Screening Methods

• Bacterial two-hybrid or split-ubiquitin membrane yeast two-hybrid systems are particularly suitable for membrane proteins like Rv3691.

• Proximity-dependent biotin identification (BioID) or APEX2-based proximity labeling can capture transient or weak interactions in their native cellular context.

• Co-immunoprecipitation followed by mass spectrometry (Co-IP-MS) using the His-tagged recombinant Rv3691 as bait can identify stable interaction partners.

Targeted Validation Approaches

• Surface plasmon resonance (SPR) or microscale thermophoresis (MST) using purified recombinant Rv3691 to measure binding affinity and kinetics with candidate interaction partners.

• Fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) to visualize interactions in live cells.

• Co-crystallization or cryo-EM studies of Rv3691 with interacting partners to determine structural details of the complexes.

How can structural studies be designed for membrane proteins like Rv3691?

Structural characterization of membrane proteins remains challenging but is essential for understanding function. For Rv3691, a multi-technique approach would be most effective:

Sample Preparation Optimization

• Expression system selection: While E. coli systems are documented for Rv3691 , eukaryotic systems like yeast might provide better folding for structural studies.

• Construct design: Creating truncated versions or specific domains might improve crystallization prospects. Analysis of the full 381-amino acid sequence can guide rational construct design.

• Detergent screening: Systematic testing of different detergents and lipid compositions to identify conditions that maintain native-like structure while allowing for structural studies.

Hybrid Structural Approaches

• X-ray crystallography: Focus on identifying stabilizing mutations or antibody fragments that can facilitate crystallization.

• Cryo-EM: Particularly suitable if Rv3691 forms larger complexes or can be incorporated into nanodiscs or lipid environments.

• Solution NMR: May be applicable for specific domains or regions of the protein that can be expressed separately.

• Integrative structural biology: Combining lower-resolution techniques (small-angle X-ray scattering, cross-linking mass spectrometry) with computational modeling to generate structural models.

How should researchers interpret contradictory data regarding Rv3691 function?

When working with uncharacterized proteins like Rv3691, researchers often encounter seemingly contradictory results from different experimental approaches. A systematic framework for resolving such contradictions includes:

Data Validation Process

• Technical validation: Confirm that assays are functioning as expected using appropriate positive and negative controls.

• Biological context consideration: Different growth conditions, strain backgrounds, or expression levels can significantly impact results. The genetic background of the H37Rv strain being used is particularly important given the documented genomic variations between laboratory stocks .

• Methodological limitations assessment: Each technique has inherent biases and limitations. For example, recombinant expression in E. coli versus yeast may yield proteins with different post-translational modifications or folding characteristics.

Integrative Data Analysis

• Hierarchical evidence weighting: Assign different weights to various types of evidence based on their directness and reliability.

• Biological plausibility assessment: Evaluate contradictory findings in the context of what is known about membrane proteins in mycobacteria and bacterial physiology.

• Alternative hypothesis generation: Consider whether contradictory data might actually indicate multiple functions or context-dependent roles for Rv3691.

What bioinformatic tools can help predict the function of Rv3691?

Given the uncharacterized nature of Rv3691, computational approaches can generate testable hypotheses about its function. A comprehensive bioinformatic analysis would include:

Sequence-Based Analysis

• Sensitive homology detection using position-specific scoring matrices, hidden Markov models, and profile-profile comparisons to identify distant relationships to characterized proteins.

• Protein family classification through comparison with specialized databases for membrane proteins and transporters.

• Genomic context analysis examining conservation of gene neighborhood across mycobacterial species to identify functionally related genes.

• Coevolution analysis to identify residues that might be functionally coupled, potentially indicating interaction interfaces or mechanistically important regions.

Structure-Based Prediction

• Ab initio structure prediction using AlphaFold2 or RosettaFold, which have shown impressive results even for membrane proteins.

• Structure-based function prediction using tools that compare predicted structural features to databases of known protein functions.

• Molecular docking simulations to explore potential binding partners or substrates based on structural predictions.

• Molecular dynamics simulations to study dynamics and conformational changes that might provide functional insights.

What are the major challenges in working with recombinant membrane proteins like Rv3691?

Working with membrane proteins presents unique technical challenges compared to soluble proteins. For Rv3691, researchers should anticipate and address the following issues:

Expression and Purification Challenges

• Protein toxicity: Overexpression of membrane proteins often causes toxicity to host cells, limiting yield. The use of tightly controlled inducible promoters and specialized host strains can mitigate this issue.

• Inclusion body formation: Membrane proteins frequently aggregate in inclusion bodies when overexpressed. While E. coli expression systems have been used successfully for Rv3691 , optimization of induction conditions (temperature, inducer concentration, duration) is critical.

• Extraction efficiency: The choice of detergent significantly impacts extraction efficiency and protein stability. Systematic screening of detergent types and concentrations is necessary to optimize purification protocols.

• Protein stability: Maintaining the stability of purified Rv3691 requires careful buffer formulation. The inclusion of stabilizing agents such as trehalose (6%) and glycerol in storage buffers helps preserve protein integrity.

Functional Assay Development

• Native environment reconstitution: Designing assays that mimic the native membrane environment is essential for accurate functional characterization. Options include reconstitution into liposomes, nanodiscs, or detergent micelles.

• Functional readout selection: Without prior knowledge of function, selecting appropriate readouts for activity assays is challenging. A multi-pronged approach measuring binding, conformational changes, and potential enzymatic activities may be necessary.

• Assay validation: Confirming that in vitro assays reflect in vivo function requires careful controls and complementary approaches, such as genetic validation in mycobacterial systems.

How can researchers overcome solubility and stability issues with Rv3691?

Maintaining solubility and stability of membrane proteins is critical for successful structural and functional studies. For Rv3691, specific strategies include:

Solubility Enhancement Strategies

• Fusion tags beyond the standard His-tag can improve solubility. Options include MBP (maltose-binding protein), SUMO, or Mistic fusions specifically designed for membrane proteins.

• Detergent screening should be systematic and include newer amphipathic agents like SMA copolymers that can extract membrane proteins with their surrounding lipids.

• Lipid supplementation during extraction and purification can stabilize native-like conformations. Mycobacterial membrane lipids or synthetic lipid mixtures mimicking mycobacterial membranes may be particularly effective.

Stability Optimization Approaches

• Buffer optimization beyond standard conditions. The reported Tris/PBS-based buffer with 6% trehalose at pH 8.0 provides a starting point, but systematic screening of pH, salt concentration, and additives should be performed.

• Thermostability assays such as differential scanning fluorimetry can rapidly identify stabilizing conditions for subsequent functional or structural studies.

• Protein engineering approaches including surface entropy reduction, disulfide engineering, or the identification of stabilizing mutations through directed evolution can generate more stable variants for characterization.