Recombinant Tolumonas auensis UPF0114 protein Tola_1474 (Tola_1474)

What is Tolumonas auensis UPF0114 protein Tola_1474 and what are its basic properties?

Tola_1474 is a 167-amino acid protein belonging to the UPF0114 protein family, derived from Tolumonas auensis, a gram-negative bacterium originally isolated from anoxic sediments of a freshwater lake. The recombinant form is typically expressed with an N-terminal His tag to facilitate purification and subsequent experimental applications .

The protein has several notable characteristics:

• Full amino acid sequence: MERFIERLMYSARWIMAPIYLGLSLALLALGIKFFQEVFHIFTVIISMKEVELILIILSLIDISLVGGLIVMVMYSGYENFVSRLDLDDHDDKLSWLGKLDAGSLKNKVAASIVAISSIHLLKVFMNTENIADDKIKWYLLIHITFVMSAFAMGYLDKLLRDKDSPH

• Protein length: 167 amino acids (full-length)

• UniProt ID: C4LES1

• Analysis of the sequence suggests the presence of multiple transmembrane regions, consistent with a membrane-associated function

What is known about the source organism Tolumonas auensis?

Tolumonas auensis (strain TA4) is a unique bacterium with several distinctive properties:

• It was isolated from anoxic sediments of a freshwater lake

• Cells are nonmotile, gram-negative rods measuring 0.9 to 1.2 by 2.5 to 3.2 microns

• Optimal growth conditions are 22°C and pH 7.2

• DNA G+C content: 49 mol%

• It has the unusual capability of producing toluene from phenylalanine, phenylpyruvate, phenyllactate, and phenylacetate

• It can also produce phenol from tyrosine

• Growth occurs under both oxic and anoxic conditions, demonstrating metabolic versatility

• Major fermentation products when grown on glucose include acetate, ethanol, and formate

• Contains ubiquinone 8 and menaquinone 8 as major lipoquinones under both oxic and anoxic growth conditions

• Taxonomically classified in the gamma subclass of Proteobacteria based on 16S ribosomal DNA sequence analysis

What are the recommended storage and handling conditions for recombinant Tola_1474?

For optimal stability and activity of recombinant Tola_1474, follow these methodological guidelines:

• Initial receipt handling:

  • The protein is typically supplied as a lyophilized powder

  • Briefly centrifuge the vial before opening to bring contents to the bottom

• Storage conditions:

  • Unopened: Store at -20°C to -80°C upon receipt

  • Working aliquots: Can be maintained at 4°C for up to one week

• Reconstitution protocol:

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being standard in many laboratories)

  • Aliquot to avoid repeated freeze-thaw cycles

• Buffer considerations:

  • Storage buffer typically consists of Tris/PBS-based buffer with 6% Trehalose at pH 8.0

  • This formulation helps maintain protein stability during freeze-thaw cycles

What expression systems are optimal for recombinant Tola_1474 production?

Based on available research data, E. coli has been successfully employed as the primary expression system for recombinant Tola_1474 . A methodological approach to expression optimization includes:

• Expression vector selection:

  • Vectors containing T7 or tac promoters for inducible expression

  • Integration of an N-terminal His tag for affinity purification

  • Consideration of fusion partners (e.g., MBP, SUMO) if expression yields are suboptimal

• E. coli strain optimization:

  • BL21(DE3) and derivatives are commonly used for recombinant protein expression

  • For membrane-associated proteins like Tola_1474, specialized strains such as C41(DE3) or C43(DE3) may offer advantages

• Induction parameters to systematically optimize:

  • IPTG concentration: Typically 0.1-1.0 mM

  • Induction temperature: Lower temperatures (16-25°C) often yield better solubility for membrane-associated proteins

  • Induction duration: 4-18 hours, with extended times at lower temperatures

• Cell lysis and protein extraction:

  • For membrane-associated proteins, inclusion of detergents may be necessary

  • Gentle lysis methods (e.g., enzymatic lysis with lysozyme followed by mild sonication) may preserve protein structure

What purification strategy would yield highest purity Tola_1474 for structural studies?

A multi-step purification approach is recommended for obtaining high-purity Tola_1474 suitable for structural and functional studies:

• Immobilized Metal Affinity Chromatography (IMAC):

  • Primary capture using Ni-NTA or Co-NTA resins

  • Binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10-20 mM imidazole

  • Wash buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20-50 mM imidazole

  • Elution buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250-300 mM imidazole

• Size Exclusion Chromatography (SEC):

  • Secondary purification step to remove aggregates and further purify the protein

  • Recommended column: Superdex 75 or Superdex 200

  • Running buffer: 20 mM Tris-HCl pH 8.0, 150 mM NaCl

• Quality control assessments:

  • SDS-PAGE analysis to confirm >90% purity

  • Western blotting with anti-His antibodies to verify identity

  • Mass spectrometry to confirm molecular weight and sequence integrity

How can researchers troubleshoot low yield or poor solubility of recombinant Tola_1474?

When encountering challenges with Tola_1474 expression and purification, apply these methodological approaches:

• For low expression yields:

  • Optimize codon usage for E. coli

  • Reduce expression temperature (16-20°C)

  • Test different E. coli host strains

  • Consider auto-induction media instead of IPTG induction

  • Evaluate expression at different time points post-induction

• For poor solubility:

  • Include membrane-mimetic environments during extraction:

    • Detergents (DDM, LDAO, Triton X-100)

    • Lipid nanodiscs

    • Amphipols

  • Modify lysis buffer components:

    • Add glycerol (5-10%)

    • Include mild stabilizing agents

    • Optimize salt concentration (150-500 mM)

• For protein instability:

  • Add protease inhibitors during all purification steps

  • Minimize purification time

  • Maintain samples at 4°C throughout processing

  • Test different pH conditions (pH 7.0-8.5)

  • Consider additives such as trehalose or sucrose

What computational methods can predict the structure and function of Tola_1474?

A systematic computational approach to Tola_1474 structural and functional prediction includes:

• Sequence-based analyses:

  • Secondary structure prediction using PSIPRED or JPred

  • Transmembrane topology prediction with TMHMM or Phobius

  • Identification of conserved domains and motifs using InterPro or Pfam

  • Analysis of sequence conservation patterns across homologs

• Three-dimensional structure prediction:

  • Template-based modeling if homologous structures exist

  • Ab initio modeling using Rosetta or I-TASSER

  • Deep learning approaches like AlphaFold2 or RoseTTAFold

  • Molecular dynamics simulations to refine predictions

• Functional annotation:

  • Gene ontology term prediction

  • Protein-protein interaction network analysis

  • Metabolic pathway mapping

  • Comparison with functionally characterized homologs

• Validation of predictions:

  • Comparison across different prediction methods

  • Assessment of prediction confidence scores

  • Experimental validation of key predictions through targeted mutagenesis

What experimental approaches can determine the membrane topology of Tola_1474?

Given the predicted membrane association of Tola_1474, these methodological approaches can experimentally determine its topology:

• Cysteine accessibility methods:

  • Substitute native cysteines with alanine

  • Introduce single cysteines at strategic positions

  • Probe accessibility with membrane-permeable and impermeable thiol-reactive reagents

  • Analysis by mass spectrometry or gel mobility shifts

• Protease protection assays:

  • Express Tola_1474 in membranous systems

  • Treat with proteases in the presence and absence of membrane permeabilization

  • Identify protected fragments by Western blotting or mass spectrometry

  • Map results to determine membrane-embedded regions

• Fluorescence-based approaches:

  • GFP fusion analysis to terminal and internal sites

  • pH-sensitive fluorescent protein tags to determine luminal/cytoplasmic orientation

  • FRET-based distance measurements between domains

• Structural biology techniques:

  • Cryo-electron microscopy of membrane-embedded protein

  • X-ray crystallography of detergent-solubilized or lipid cubic phase preparations

  • Solid-state NMR of isotopically labeled protein in membrane mimetics

How can researchers design experiments to elucidate the function of Tola_1474?

A comprehensive strategy for functional characterization includes:

• Gene knockout/complementation studies:

  • Generate Tola_1474 deletion mutants in Tolumonas auensis

  • Characterize phenotypic changes under various growth conditions

  • Complement with wild-type and mutant versions to confirm specificity

  • Monitor effects on toluene production pathways

• Protein-protein interaction studies:

  • Pull-down assays using His-tagged Tola_1474

  • Bacterial two-hybrid screening

  • Cross-linking coupled with mass spectrometry

  • Co-immunoprecipitation with candidate interacting partners

• Localization studies:

  • Immunogold electron microscopy

  • Fluorescent protein fusions with live-cell imaging

  • Subcellular fractionation and Western blotting

  • Super-resolution microscopy for detailed localization patterns

• Functional assays based on predicted roles:

  • If transport function is suspected:

    • Liposome reconstitution with transport assays

    • Membrane potential measurements

    • Substrate binding studies

  • If enzymatic activity is predicted:

    • Activity assays with potential substrates

    • Metabolite profiling in wild-type vs. knockout strains

How can site-directed mutagenesis be applied to study Tola_1474 structure-function relationships?

A systematic mutagenesis approach provides insights into critical functional elements:

• Strategic target selection:

  • Conserved residues across UPF0114 family members

  • Predicted transmembrane regions and interface residues

  • Potential substrate binding or catalytic sites

  • Charged residues within transmembrane domains

• Experimental design table for site-directed mutagenesis:

• Mutation validation workflow:

  • Verify expression levels comparable to wild-type

  • Confirm proper folding and stability

  • Assess subcellular localization

  • Quantify functional changes with appropriate assays

  • Determine effects on protein-protein interactions

How can researchers investigate potential roles of Tola_1474 in Tolumonas auensis' toluene production pathway?

Given the unique toluene-producing capability of Tolumonas auensis, methodological approaches to investigate Tola_1474's potential involvement include:

• Comparative expression analysis:

  • Quantify Tola_1474 expression under conditions that induce/repress toluene production

  • Compare expression patterns with known enzymes in the toluene synthesis pathway

  • Analyze co-expression networks to identify functional relationships

• Metabolic impact studies:

  • Generate Tola_1474 knockout strains

  • Measure toluene production from precursors (phenylalanine, phenylpyruvate)

  • Conduct metabolic flux analysis using labeled precursors

  • Identify accumulating intermediates or altered byproducts

• Protein interaction studies:

  • Screen for interactions with enzymes in the toluene synthesis pathway

  • Test for binding of pathway intermediates or regulators

  • Investigate potential roles in transport of precursors or products

• Structural modeling focused on potential roles:

  • Model potential binding sites for aromatic compounds

  • Compare structural features with known transporters or enzymes

  • Design mutations to test computational predictions

What advanced imaging techniques can provide insights into Tola_1474 function in situ?

Advanced imaging methodologies can reveal spatial and temporal aspects of Tola_1474 function:

• Super-resolution microscopy approaches:

  • Stimulated Emission Depletion (STED) microscopy

  • Photoactivated Localization Microscopy (PALM)

  • Single-Molecule Localization Microscopy (SMLM)

  • These techniques achieve resolution below the diffraction limit (typically 20-50 nm)

• Single-molecule imaging strategies:

  • Single-particle tracking of fluorescently labeled Tola_1474

  • Analysis of diffusion coefficients in different cellular regions

  • Determination of residence times at specific cellular locations

  • Detection of potential oligomerization events

• Correlative microscopy methods:

  • Correlative Light and Electron Microscopy (CLEM)

  • Combines fluorescence localization with ultrastructural context

  • Provides nanoscale resolution of protein distribution relative to cellular ultrastructure

• Dynamic imaging approaches:

  • Fluorescence Recovery After Photobleaching (FRAP)

  • Förster Resonance Energy Transfer (FRET)

  • Fluorescence Lifetime Imaging (FLIM)

  • These techniques can reveal protein dynamics and interactions in living cells

How should researchers design experiments to distinguish between multiple potential functions of Tola_1474?

When investigating a protein with unclear function like Tola_1474, employ these methodological approaches:

• Parallel hypothesis testing framework:

  • Formulate multiple function hypotheses based on bioinformatic predictions

  • Design specific experiments to test each hypothesis

  • Implement positive and negative controls for each functional assay

  • Apply Bayesian analysis to update hypothesis probabilities as data accumulates

• Multi-level experimental design:

  • In silico: Computational predictions and modeling

  • In vitro: Purified protein functional assays

  • In vivo: Cellular localization and knockout studies

  • Systems-level: Transcriptomic and metabolomic impacts

• Orthogonal validation approach:

  • Test each potential function using multiple independent methods

  • Triangulate results across different experimental approaches

  • Identify consistent patterns that emerge across diverse techniques

• Decision tree for sequential experimentation:

How can researchers analyze complex datasets from Tola_1474 functional studies?

Modern data analysis approaches for complex protein characterization include:

• Integrative data analysis workflow:

  • Data quality assessment and preprocessing

  • Normalization appropriate to each data type

  • Statistical testing with proper controls for multiple comparisons

  • Integration of multiple data types using computational frameworks

• Statistical approaches for different experimental designs:

  • For comparing mutants: ANOVA with post-hoc tests

  • For dose-response relationships: Non-linear regression modeling

  • For time-series data: Mixed-effects models or functional data analysis

  • For omics data: Multivariate methods (PCA, clustering)

• Machine learning applications:

  • Supervised learning to classify protein functions

  • Unsupervised learning to identify patterns in complex datasets

  • Feature selection to identify key experimental variables

  • Model validation through cross-validation and external validation sets

• Biological network analysis:

  • Integration of protein-protein interaction data

  • Pathway enrichment analysis

  • Network visualization and community detection

  • Identification of functional modules and potential regulatory relationships

How can contradictory results in Tola_1474 research be reconciled methodologically?

When faced with contradictory results, apply these systematic resolution approaches:

• Methodological reconciliation workflow:

  • Carefully document all experimental conditions and variables

  • Identify specific points of contradiction between studies

  • Design experiments specifically addressing contradictory elements

  • Implement standardized protocols across research groups

• Sources of variation to investigate:

  • Protein construct differences (tags, fusion partners)

  • Expression systems and purification methods

  • Buffer compositions and experimental conditions

  • Detection methods and their sensitivity/specificity

  • Statistical power and sample sizes

• Collaborative validation approaches:

  • Inter-laboratory validation studies

  • Sharing of reagents and standardized protocols

  • Blind testing of key hypotheses

  • Meta-analysis of compiled data across studies

• Embracing biological complexity:

  • Consider context-dependent protein functions

  • Investigate condition-specific effects

  • Explore potential post-translational modifications

  • Evaluate impacts of interaction partners or membrane environment

What emerging technologies could advance understanding of Tola_1474 function?

Several cutting-edge technologies offer promising approaches for deeper insights:

• Cryo-electron microscopy advances:

  • Single-particle analysis for high-resolution structure determination

  • Cryo-electron tomography for in situ structural visualization

  • Time-resolved cryo-EM for capturing conformational dynamics

• Integrative structural biology approaches:

  • Combining multiple structural techniques (X-ray, NMR, SAXS)

  • Integrative modeling using sparse and diverse experimental data

  • In-cell structural determination using emerging methods

• Advanced genetic tools:

  • CRISPR interference for tunable gene expression control

  • Base editing for precise point mutations

  • Proximity labeling for in vivo interaction mapping

  • Optogenetic control of protein activity

• Systems biology frameworks:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Genome-scale metabolic modeling

  • Protein structure and function prediction using deep learning

How might research on Tola_1474 contribute to understanding bacterial membrane protein evolution?

Tola_1474 research offers opportunities to address fundamental questions in protein evolution:

• Comparative genomics approaches:

  • Identify UPF0114 family members across bacterial lineages

  • Analyze sequence conservation patterns and selection pressures

  • Reconstruct evolutionary history and potential functional divergence

  • Identify co-evolving gene families that may indicate functional relationships

• Structure-guided evolutionary analysis:

  • Map conservation onto structural models

  • Identify structurally conserved but sequence-variable regions

  • Analyze evolution of transmembrane topology

  • Compare with other membrane protein families

• Experimental evolution studies:

  • Direct evolution experiments under selective pressures

  • Analysis of mutational tolerance across protein regions

  • Investigation of potential adaptive mutations

  • Reconstruction of ancestral protein sequences

• Functional innovation exploration:

  • Investigate functional diversity across UPF0114 family members

  • Test for neofunctionalization or subfunctionalization

  • Analyze contributions to species-specific metabolic capabilities

  • Connect to Tolumonas auensis' unique toluene production ability

What potential biotechnological applications might emerge from Tola_1474 research?

Understanding this protein could lead to several applied outcomes:

• Biocatalysis applications:

  • If enzymatic activity is discovered, potential use in biocatalytic processes

  • Engineering of enzyme variants with enhanced activity or substrate specificity

  • Application in biosynthesis of aromatic compounds

  • Potential roles in bioremediation of aromatic pollutants

• Membrane protein engineering platforms:

  • Development as a scaffold for engineered membrane proteins

  • Creation of chimeric proteins with novel functions

  • Template for computational design of membrane proteins

  • Model system for studying membrane protein folding and stability

• Biosensor development:

  • If substrate binding capabilities are identified, potential biosensor applications

  • Development of whole-cell biosensors for environmental monitoring

  • Creation of protein-based detection systems for specific compounds

• Metabolic engineering applications:

  • Potential roles in engineered pathways for aromatic compound production

  • Contribution to biofuel or fine chemical biosynthesis

  • Enhancement of toluene production capabilities in industrial strains