Recombinant Cupriavidus pinatubonensis UPF0060 membrane protein Reut_B3679 (Reut_B3679)

What is Recombinant Cupriavidus pinatubonensis UPF0060 membrane protein Reut_B3679?

Recombinant Cupriavidus pinatubonensis UPF0060 membrane protein Reut_B3679 (UniProt ID: Q46UZ7) is a membrane-associated protein derived from the bacterial strain Cupriavidus pinatubonensis (strain JMP134/LMG 1197), previously known as Alcaligenes eutrophus or Ralstonia eutropha. The protein belongs to the UPF0060 protein family, which consists of uncharacterized membrane proteins with predicted transmembrane domains. The amino acid sequence indicates a highly hydrophobic protein with multiple potential membrane-spanning regions: MNTIALYLLTAVAEILGCYLPYLWLRQGASAWVLLPGALSLALFAWLLSLHPDASGRVYAAYGGVYIGVAVLWLWLVDGVRPSAWDLAGVGVAFGGMAIIV .

How does Reut_B3679 compare structurally to other membrane proteins in Cupriavidus species?

Reut_B3679 shares structural characteristics with other bacterial membrane proteins, particularly those involved in transmembrane transport and signaling. Analysis of its sequence reveals multiple hydrophobic regions consistent with transmembrane domains, which likely anchor the protein within the bacterial membrane. Unlike the better-characterized uridylate kinase (pyrH) from the same organism , Reut_B3679 lacks well-defined enzymatic domains but demonstrates the classic alternating hydrophobic-hydrophilic pattern typical of membrane-spanning proteins. Structural prediction indicates similarities to proteins involved in the endoplasmic reticulum membrane protein complex (EMC) seen in eukaryotes, which facilitate the biogenesis of multipass transmembrane proteins .

What are the predicted functional domains of Reut_B3679 and their significance in research?

Sequence analysis of Reut_B3679 reveals several key functional regions:

These domains suggest Reut_B3679 may function in membrane transport, signaling, or structural support. Research significance lies in understanding how these bacterial membrane proteins compare to more complex eukaryotic membrane protein systems, potentially providing insights into fundamental mechanisms of membrane protein biogenesis and function .

What are the optimal conditions for studying Reut_B3679 in vitro?

When designing experiments to study Reut_B3679, researchers should consider the following optimal conditions:

• Storage: The recombinant protein should be stored in Tris-based buffer with 50% glycerol at -20°C for short-term use or -80°C for extended storage. Avoid repeated freeze-thaw cycles, and working aliquots can be maintained at 4°C for up to one week .

• Reconstitution: Due to its highly hydrophobic nature, Reut_B3679 requires careful handling during reconstitution. Mild detergents such as n-dodecyl-β-D-maltoside (DDM) at concentrations just above critical micelle concentration (CMC) are recommended for solubilization while preserving protein structure.

• Experimental buffers: Phosphate buffers (pH 7.0-7.5) supplemented with stabilizing agents are most effective for maintaining protein stability during functional assays.

These recommendations align with standard approaches for membrane protein research while accounting for the specific properties of Reut_B3679 based on its amino acid composition and predicted membrane-spanning regions .

How should I design experiments to investigate potential interactions between Reut_B3679 and other membrane proteins?

When investigating potential protein-protein interactions involving Reut_B3679, consider the following experimental design principles:

• Co-immunoprecipitation approach: Design experiments using tagged versions of Reut_B3679 (such as FLAG-tagged constructs) to pull down potential interaction partners. This approach has been successfully employed with other membrane proteins like Emc3-3xFLAG to identify interactions with multipass membrane proteins and chaperones .

• Proximity-based labeling: Implement BioID or APEX2-based proximity labeling by fusing these enzymes to Reut_B3679 to identify proteins in close proximity within the membrane environment.

• Controls and validation: Include proper controls such as unrelated membrane proteins (like Orm1 in yeast studies) to distinguish specific from non-specific interactions . Quantitative approaches like SILAC can provide statistical confidence in identified interactions.

• Variables to control: When designing these experiments, control for:

  • Detergent concentration and type

  • Salt concentration in buffers

  • Temperature during solubilization

  • Expression levels of recombinant proteins

These approaches draw on successful strategies used for other membrane proteins while accounting for the specific challenges posed by UPF0060 family proteins .

What experimental variables should be controlled when assessing the impact of Reut_B3679 on bacterial membrane integrity?

When assessing the impact of Reut_B3679 on bacterial membrane integrity, researchers should control the following variables:

The experimental design should include appropriate controls (non-expressing strains, expression of unrelated membrane proteins) and multiple complementary assays to evaluate membrane integrity, such as fluorescent dye permeability tests, electron microscopy, and membrane potential measurements. This multi-faceted approach aligns with general experimental design principles while tailoring the specific variables to membrane protein biology .

How can I utilize Reut_B3679 to study bacterial membrane protein biogenesis pathways?

Reut_B3679 can serve as an excellent model for studying bacterial membrane protein biogenesis due to its multiple transmembrane domains and uncharacterized function. Researchers can leverage this protein through several approaches:

• Cotranslational insertion studies: Similar to studies with the ER membrane protein complex (EMC), researchers can employ proximity-specific ribosome profiling to identify when Reut_B3679 engages with membrane insertion machinery during translation . This technique involves crosslinking nascent chains to nearby factors during synthesis and sequencing the associated mRNA fragments.

• Chaperone interaction network mapping: By performing systematic pulldown experiments with tagged Reut_B3679 under various conditions (normal growth, stress conditions, etc.), researchers can identify the suite of chaperones that facilitate its proper folding and insertion, similar to approaches used with EMC clients .

• TMD analysis experiments: The charged and bulky residues within Reut_B3679's transmembrane domains make it an excellent candidate for studying how "challenging" TMDs are processed by bacterial insertion machinery. Systematic mutagenesis of these residues, coupled with folding and localization assays, can provide insights into the biogenesis pathways for difficult-to-fold membrane proteins .

These approaches build upon established methodologies while focusing on the unique properties of Reut_B3679, providing a framework for understanding fundamental aspects of bacterial membrane protein biogenesis.

What methodologies can be employed to investigate the role of Reut_B3679 in bacterial stress response?

To investigate potential roles of Reut_B3679 in bacterial stress response, researchers should implement the following methodologies:

• Gene knockout and complementation studies: Generate knockout strains (ΔReut_B3679) and complemented strains, then expose them to various stresses (oxidative, osmotic, pH, temperature) to evaluate changes in survival, growth rates, and membrane integrity.

• Transcriptional profiling under stress conditions: Employ RNA-seq or microarray analysis comparing wild-type and ΔReut_B3679 strains under normal and stress conditions to identify transcriptional networks affected by the absence of this protein.

• Proteomics-based interactome analysis: Use crosslinking mass spectrometry (XL-MS) or co-immunoprecipitation coupled with LC-MS/MS to identify proteins that interact with Reut_B3679 specifically under stress conditions, similar to approaches used with other membrane protein complexes .

• In vivo localization studies: Utilize fluorescent protein fusions to track changes in Reut_B3679 localization during stress response, providing insights into potential redistribution or clustering within the membrane.

These methodologies should be implemented using a systematic experimental design approach with appropriate controls and replicates to ensure reliable data interpretation . The combination of genetic, transcriptomic, proteomic, and imaging approaches provides a comprehensive view of Reut_B3679's potential role in stress response.

How can I design experiments to elucidate the potential role of Reut_B3679 in bacterial membrane transport?

To investigate Reut_B3679's potential role in membrane transport, implement a comprehensive experimental approach:

• Substrate transport assays: Design experiments measuring the uptake or efflux of radioactively labeled or fluorescent substrates in wild-type versus ΔReut_B3679 strains. Select potential substrates based on structural similarities to known transporters with comparable transmembrane domain arrangements.

• Membrane reconstitution experiments: Purify Reut_B3679 and reconstitute it into liposomes with defined composition. Measure transport of candidate substrates across these proteoliposomes using:

  • Fluorescence-based assays with substrate-sensitive dyes

  • Isotope flux measurements

  • Electrical measurements for charged substrate transport

• Structure-function analysis: Implement systematic mutagenesis focusing on:

  • Charged residues within transmembrane domains (particularly regions similar to those found in transporters)

  • Potential substrate binding sites identified through computational modeling

  • Conserved motifs identified through comparative sequence analysis

• Biophysical characterization: Employ techniques such as:

  • Microscale thermophoresis to measure substrate binding

  • Hydrogen-deuterium exchange mass spectrometry to identify conformational changes upon substrate binding

  • Cryo-EM to determine structural features in different functional states

This multi-faceted approach combines genetic, biochemical, and biophysical methods, drawing on established protocols for membrane transport proteins while accounting for the specific challenges and properties of Reut_B3679 .

What are common challenges in expressing and purifying Reut_B3679, and how can they be addressed?

Researchers working with Reut_B3679 commonly encounter several challenges during expression and purification:

These challenges reflect the general difficulties in membrane protein biochemistry but are particularly relevant for Reut_B3679 given its multiple transmembrane domains and the presence of charged residues within those domains . The solutions draw on successful approaches with other challenging membrane proteins while specifically addressing the properties of UPF0060 family proteins.

How can I troubleshoot non-specific binding issues when studying Reut_B3679 interactions with other proteins?

When troubleshooting non-specific binding issues in Reut_B3679 interaction studies:

• Optimize detergent conditions: Test a gradient of detergent concentrations to find the minimal concentration that maintains Reut_B3679 solubility while reducing non-specific hydrophobic interactions. Consider detergent exchange during purification to milder detergents.

• Implement proper controls: Use unrelated membrane proteins of similar size and hydrophobicity (such as Orm1 used in EMC studies) as negative controls to distinguish specific from non-specific interactions.

• Adjust buffer conditions systematically:

  • Increase salt concentration incrementally (50-500mM) to disrupt electrostatic interactions

  • Test different pH conditions that maintain protein stability

  • Add low concentrations of competitive agents like reduced amino acids or mild chaotropes

• Utilize quantitative approaches: Implement SILAC or other quantitative proteomics approaches to statistically differentiate true interactors from background, as demonstrated in studies with Emc3-3xFLAG .

• Consider crosslinking optimization: If using crosslinking approaches, perform a detailed optimization of crosslinker type, concentration, and reaction time to capture specific interactions while minimizing random crosslinking events.

These troubleshooting approaches draw on established principles in membrane protein biochemistry while addressing the specific challenges posed by Reut_B3679's membrane localization and potentially dynamic interaction network .

What data analysis challenges might arise when interpreting results from Reut_B3679 functional studies?

Researchers performing functional studies with Reut_B3679 should anticipate several data analysis challenges:

• Differentiating direct from indirect effects: When phenotyping ΔReut_B3679 strains, observed changes may result from indirect effects due to altered membrane properties rather than direct loss of protein function. Address this by:

  • Implementing complementation studies with wild-type and mutant variants

  • Comparing effects with knockouts of functionally related proteins

  • Conducting time-course analyses to distinguish primary from secondary effects

• Accounting for variable expression levels: Variation in expression levels between experiments can confound functional interpretations. Implement:

  • Internal normalization standards

  • Quantitative Western blotting to correlate function with expression level

  • Statistical methods that account for expression-level dependencies

• Interpreting complex membrane interaction networks: As seen with eukaryotic membrane protein complexes like the EMC , membrane proteins often function within complex interaction networks. Address this complexity by:

  • Using network analysis approaches to visualize and interpret interaction data

  • Implementing conditional knockout strategies to dissect contribution of individual components

  • Developing mathematical models that account for the combinatorial effects of multiple interactions

• Controlling for membrane perturbation artifacts: Overexpression or deletion of membrane proteins can disrupt membrane homeostasis, creating non-specific effects. Control for this by:

  • Measuring general membrane parameters in all experimental conditions

  • Including membrane-targeted controls that are not expected to have specific functional effects

  • Titrating expression levels to determine dose-dependent versus threshold effects

How might studying Reut_B3679 contribute to our understanding of membrane protein evolution?

Studying Reut_B3679 offers several opportunities to advance our understanding of membrane protein evolution:

• Evolutionary conservation analysis: The UPF0060 protein family represents an uncharacterized group of membrane proteins conserved across diverse bacterial species. Comparative genomic analyses of Reut_B3679 homologs can reveal evolutionary patterns in membrane protein conservation, particularly focusing on:

  • Conservation of charged residues within transmembrane domains

  • Co-evolution with interacting proteins

  • Horizontal gene transfer patterns across bacterial phyla

• Structural homology with ancient membrane protein families: Reut_B3679 may share structural features with evolutionarily ancient membrane protein families. Similar to EMC3's proposed shared ancestry with the YidC/Oxa1/Alb3 protein family , Reut_B3679 might represent an evolutionary link between different membrane protein insertion or chaperoning systems.

• Functional adaptation across ecological niches: Cupriavidus pinatubonensis is known for its metabolic versatility and ability to degrade aromatic compounds. Comparing Reut_B3679 variants across Cupriavidus species adapted to different environments could reveal how membrane proteins evolve in response to ecological pressures.

These evolutionary analyses can provide insights into fundamental principles of membrane protein evolution, potentially revealing how complex membrane protein systems in higher organisms evolved from simpler bacterial predecessors .

What potential applications might Reut_B3679 have in synthetic biology and bioengineering?

Reut_B3679 presents several promising applications for synthetic biology and bioengineering:

• Membrane protein expression platform: The structural features of Reut_B3679 could be exploited to develop improved expression systems for difficult-to-express membrane proteins. By understanding how Reut_B3679 successfully integrates into the membrane despite challenging features (charged residues in TMDs), researchers could engineer expression systems that facilitate the production of therapeutically important mammalian membrane proteins.

• Biosensor development: Reut_B3679's multiple transmembrane domains make it a potential scaffold for developing membrane-embedded biosensors. By engineering substrate-binding domains or integrating reporter elements, researchers could create sensors for environmental monitoring or metabolic engineering applications.

• Synthetic membrane protein complexes: Drawing inspiration from natural membrane protein complexes like the EMC , engineered variants of Reut_B3679 could serve as building blocks for synthetic membrane protein complexes with novel functions, such as:

  • Controlled transport of non-native molecules

  • Artificial signaling pathways spanning bacterial membranes

  • Modular membrane protein assembly systems

• Bioremediation enhancements: Given Cupriavidus pinatubonensis' natural capacity for degrading environmental pollutants, engineered Reut_B3679 variants could potentially enhance uptake or efflux of specific contaminants, improving bioremediation applications.

These applications represent the intersection of fundamental membrane protein research with synthetic biology approaches, potentially yielding both biotechnological innovations and deeper insights into membrane protein function .

What are the most significant unanswered questions regarding Reut_B3679 that warrant further investigation?

Several critical questions about Reut_B3679 remain unanswered and represent high-priority areas for future research:

• Physiological function: The natural biological role of Reut_B3679 in Cupriavidus pinatubonensis remains unknown. Does it function as a transporter, signaling protein, structural component, or have another role entirely in bacterial physiology?

• Structural characteristics: No high-resolution structure exists for Reut_B3679 or its homologs. Determining its structure would provide insights into how its charged transmembrane residues are accommodated within the membrane and potentially reveal functional binding sites.

• Interaction network: Similar to the complex interaction networks observed with membrane protein complexes like the EMC , identifying the full complement of proteins that interact with Reut_B3679 would provide context for understanding its cellular functions.

• Regulatory mechanisms: How is Reut_B3679 expression regulated in response to environmental conditions or cellular stress? Understanding these regulatory mechanisms could provide insights into its physiological importance.

• Evolutionary significance: As an uncharacterized protein family (UPF0060) conserved across bacterial species, understanding the evolutionary history and significance of Reut_B3679 could reveal fundamental principles of membrane protein evolution.

Addressing these questions requires integrative approaches combining structural biology, genetic manipulation, functional assays, and evolutionary analyses. The answers would not only illuminate the biology of this specific protein but could also contribute to broader understanding of bacterial membrane protein biology .

How should researchers approach integrating findings about Reut_B3679 with broader membrane protein biology concepts?

Researchers should adopt a multi-faceted approach to integrate Reut_B3679 findings with broader membrane protein biology:

• Comparative analysis framework: Establish systematic comparisons between Reut_B3679 and well-characterized membrane proteins, particularly focusing on:

  • Proteins with similar topological features

  • Proteins containing charged residues within transmembrane domains

  • Proteins with similar phylogenetic distribution

• Multi-scale integration: Connect molecular-level findings about Reut_B3679 to cellular and organismal phenotypes by:

  • Linking structural features to functional properties

  • Correlating expression patterns with physiological states

  • Developing predictive models that bridge molecular interactions and cellular outcomes

• Cross-species validation: Test hypotheses generated from Reut_B3679 studies in diverse bacterial species to distinguish universal principles from species-specific adaptations, similar to how EMC studies revealed conserved principles across yeast and human cells .

• Methodology standardization: Develop standardized protocols and reporting formats to facilitate comparison with other membrane protein studies, enabling meta-analyses and systems-level integration.

This integrative approach acknowledges that individual membrane proteins like Reut_B3679 function within complex biological systems while seeking to extract generalizable principles that advance our understanding of membrane protein biology as a whole .

What methodological innovations might accelerate research on Reut_B3679 and similar membrane proteins?

Several methodological innovations could significantly accelerate research on Reut_B3679 and similar challenging membrane proteins:

• Advanced membrane mimetics: Development of improved membrane mimetics beyond traditional detergent systems, such as:

  • Nanodiscs with tunable properties matching native bacterial membranes

  • Novel amphipathic polymers specifically designed for bacterial membrane proteins

  • Cell-free expression systems coupled with direct incorporation into membrane mimetics

• In situ structural determination: Adaptation of emerging structural biology techniques to study Reut_B3679 in its native environment:

  • Cryo-electron tomography with subtomogram averaging

  • In-cell NMR optimized for membrane proteins

  • Mass spectrometry approaches that preserve native membrane contexts

• High-throughput functional screening: Development of scalable assays to rapidly assess Reut_B3679 function:

  • Microfluidic platforms for single-cell analysis of membrane protein function

  • Multiplexed transport assays using fluorescent substrate libraries

  • Machine learning approaches to predict functional consequences of mutations

• Integrated multi-omics approaches: Similar to studies with the EMC , combining multiple omics technologies to understand Reut_B3679 in context:

  • Proximity-specific ribosome profiling to study cotranslational events

  • Spatially-resolved proteomics to map membrane protein distributions

  • Lipidomics coupled with protein analysis to understand lipid-protein interactions