Recombinant Rhizobium etli UPF0060 membrane protein RHE_CH01408 (RHE_CH01408)

What is the Rhizobium etli UPF0060 membrane protein RHE_CH01408?

The RHE_CH01408 is a membrane protein belonging to the UPF0060 family found in Rhizobium etli (strain CFN 42 / ATCC 51251), a nitrogen-fixing bacterium that forms symbiotic relationships with leguminous plants. This protein consists of 106 amino acids and is encoded by the RHE_CH01408 gene, also identified by UniProt accession number Q2KAC5 . As a membrane protein, it is characterized by hydrophobic regions suitable for membrane integration, though its precise function in nitrogen fixation processes requires further investigation.

What methodologies are recommended for expression and purification of recombinant RHE_CH01408?

For optimal expression and purification, researchers should consider:

The choice of expression system should align with downstream applications. For RHE_CH01408, commercial sources indicate that yeast expression systems have been successfully employed . Purification typically involves affinity chromatography utilizing fusion tags, followed by size-exclusion chromatography to remove aggregates. When expressing membrane proteins like RHE_CH01408, special attention must be given to detergent selection for extraction from membranes while maintaining native structure.

What quality control measures should be applied to RHE_CH01408 preparations?

Quality control for recombinant RHE_CH01408 should include:

• Identity confirmation via mass spectrometry

• Purity assessment (>90% for assay-grade proteins)

• Analytical size-exclusion chromatography to:

  • Monitor preparation quality

  • Evaluate protein stability

  • Study complex formation

  • Assess tendency to aggregate

  • Evaluate degradation profiles

For research applications requiring high structural integrity, additional quality control measures include verification of proper folding through circular dichroism and thermal stability assessment through differential scanning fluorimetry.

How does RHE_CH01408 contribute to nitrogen fixation in the Rhizobium-legume symbiosis?

While the specific function of RHE_CH01408 remains to be fully characterized, its role as a membrane protein in Rhizobium etli suggests potential involvement in critical symbiotic processes. Rhizobium etli engages in a complex developmental relationship with legumes that involves:

• Initial infection triggered by plant-derived flavonoids

• Formation of specialized nodule structures

• Bacterial differentiation into nitrogen-fixing bacteroids

As a membrane protein, RHE_CH01408 may participate in:

• Signaling pathways during symbiotic establishment

• Transport of metabolites between bacteroid and plant host

• Maintenance of membrane integrity under microaerobic conditions required for nitrogenase activity

• Facilitation of ammonia export to plant tissues

To elucidate its specific role, researchers should consider gene knockout studies within the framework of metabolic reconstruction models like iOR363, which includes 387 metabolic and transport reactions across 26 metabolic pathways in R. etli .

What approaches are recommended for structural characterization of RHE_CH01408?

Due to its membrane localization, structural characterization of RHE_CH01408 presents unique challenges. A multi-technique approach is recommended:

For crystallography, researchers should focus on generating crystallization-grade protein with verified crystallization buffers and protocols. For NMR studies, isotope labeling efficiency should be verified by mass spectrometry .

How can metabolic network modeling enhance understanding of RHE_CH01408 function?

Constraint-based metabolic modeling provides a powerful framework for investigating RHE_CH01408's role in nitrogen fixation:

• Integration into genome-scale metabolic reconstructions like iOR363

• Flux Balance Analysis (FBA) to predict metabolic flux distributions under symbiotic conditions

• In silico gene deletion studies to assess the impact on nitrogen fixation

• Comparison of model predictions with experimental observations

Research has demonstrated that FBA predictions for gene deletions during nitrogen fixation in Rhizobia align well with experimental data, suggesting that similar approaches could yield insights into RHE_CH01408 function . By simulating microaerobic conditions typical of nodule environments, researchers can predict how this membrane protein influences symbiotic nitrogen fixation activity.

What methods are optimal for studying protein-protein interactions involving RHE_CH01408?

For investigating protein-protein interactions of membrane proteins like RHE_CH01408, consider these methodologies:

When designing interaction studies, researchers should note that "the conjugation of biotin, fluorophore or other chemical entity to the protein may not affect protein structure or activity" . Site-specific conjugation strategies are therefore recommended, with careful removal of free conjugates and determination of conjugation efficiency.

How can isotope labeling of RHE_CH01408 be optimized for structural NMR studies?

For effective NMR studies of RHE_CH01408:

• Expression System Selection:

  • Bacterial systems using minimal media with 15N-ammonium salts and 13C-glucose

  • Consider specialized strains for membrane protein expression

• Labeling Strategies:

  • Uniform enrichment with 13C and 15N for 3D structure determination

  • Residue-specific labeling for focused studies of functional regions

  • Selective deuteration to improve spectral quality

• Quality Control:

  • Mass spectrometry to verify isotope incorporation efficiency

  • Test experiments to assess spectral quality

• Membrane Protein Considerations:

  • Selection of appropriate detergents or nanodiscs

  • Optimization of sample conditions for stability during extended NMR experiments

NMR spectroscopy provides valuable information about protein dynamics over various timescales through relaxation measurements, which is particularly useful for understanding membrane protein function in different environments .

How can site-directed mutagenesis elucidate functional domains of RHE_CH01408?

Site-directed mutagenesis represents a powerful approach for mapping functional regions:

• Target Selection Strategy:

  • Conserved residues identified through sequence alignment

  • Charged residues potentially involved in protein-protein interactions

  • Hydrophobic patches crucial for membrane insertion

• Mutation Design:

  • Alanine scanning to identify essential residues

  • Conservative substitutions to probe specific interactions

  • Introduction of reporter groups for localization studies

• Functional Analysis:

  • Effects on membrane localization

  • Impact on nitrogen fixation using metabolic models

  • Changes in interaction profiles with partner proteins

When integrated with the metabolic reconstruction approaches described in the literature , mutagenesis studies can provide insights into how specific residues contribute to RHE_CH01408's function in the context of symbiotic nitrogen fixation.

What challenges should researchers anticipate when working with RHE_CH01408?

As a membrane protein, RHE_CH01408 presents several technical challenges:

• Expression Difficulties:

  • Potential toxicity to host cells

  • Protein misfolding and aggregation

  • Low expression yields

• Purification Complexities:

  • Detergent selection critical for extraction and stability

  • Maintaining native conformation outside cellular environment

  • Preventing oligomerization or aggregation

• Structural Analysis Limitations:

  • Challenges in crystallization

  • Size constraints for NMR studies

  • Detergent interference with some analytical techniques

These challenges necessitate careful optimization of expression constructs, purification conditions, and analytical methods. Commercial sources of the protein may provide starting points for researchers new to this system .

How can RHE_CH01408 research contribute to agricultural applications?

Understanding RHE_CH01408's role in symbiotic nitrogen fixation has potential implications for sustainable agriculture:

• Improving Biological Nitrogen Fixation:

  • Enhanced symbiotic efficiency reducing fertilizer requirements

  • Development of more effective Rhizobium inoculants

  • Extension of symbiotic capabilities to non-legume crops

• Agricultural Impact:

  • Reduced environmental impacts from synthetic nitrogen fertilizers

  • Improved soil health through sustainable farming practices

  • Enhanced crop yields in nitrogen-limited conditions

Research into fundamental molecular mechanisms of proteins like RHE_CH01408 provides the foundation for biotechnological applications that could transform agricultural practices . The genome-scale metabolic modeling approaches described in the literature offer frameworks for predicting how modifications to these systems might enhance symbiotic nitrogen fixation.