Recombinant Escherichia coli O9:H4 UPF0060 membrane protein ynfA (ynfA)

What is the YnfA protein and what family does it belong to?

YnfA is a membrane protein belonging to the Small Multidrug Resistance (SMR) family of efflux transporters. It plays a functional role in promoting antimicrobial resistance in bacterial pathogens. The protein consists of 108 amino acids with a molecular weight of approximately 11.9 kDa and is encoded by the ynfA gene. The amino acid sequence is: MIKTTLLFFATALCEIIGCFLPWLWLKRNASIWLLLPAGISLALFVWLLTLHPAASGRVY AAYGGVYVCTALIWLRVVDGVKLTLYDWTGALIALCGMLIIVAGWGRT . As a membrane protein with transmembrane helices, YnfA typically displays anomalous migration patterns on SDS-PAGE gels, often running at an apparent molecular weight slightly higher than its calculated mass .

How is recombinant YnfA typically expressed and purified?

Recombinant YnfA is typically expressed in Escherichia coli expression systems. The full-length protein (amino acids 1-108) can be produced with an N-terminal histidine tag to facilitate purification. The recombinant protein is expressed in E. coli and purified using affinity chromatography. After purification, the protein is often provided as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE . For experimental use, the protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with addition of 5-50% glycerol (final concentration) for long-term storage at -20°C/-80°C .

What is the subcellular localization and topology of YnfA?

YnfA is an integral membrane protein that localizes to the bacterial cell membrane. Based on structural prediction studies using model transporters like EmrE, YnfA likely contains multiple transmembrane domains that span the bacterial membrane . Computational analyses have been used to predict the three-dimensional structure of YnfA, helping researchers understand its membrane topology and functional aspects as an efflux pump.

How can I confirm the expression of recombinant YnfA protein?

Expression of recombinant YnfA can be confirmed using Western blot analysis with appropriate antibodies. For His-tagged YnfA constructs, anti-His antibodies are commonly used for detection. In previous studies, YnfA expression has been confirmed using Western blot with anti-HisA antibody after expressing the protein from vectors such as pBAD_Myc_HisA . When conducting SDS-PAGE analysis, it's important to note that membrane proteins like YnfA may display gel shifts and migrate anomalously compared to soluble protein standards. YnfA, with a molecular weight of 11.9 kDa, typically appears just below the 15 kDa band of standard protein ladders . Equal total protein loading should be ensured when comparing wild-type and mutant YnfA expression levels.

What are the recommended storage conditions for maintaining YnfA protein stability?

For optimal stability, recombinant YnfA protein should be stored according to the following guidelines:

The recommended reconstitution procedure involves briefly centrifuging the vial prior to opening, then reconstituting the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL with addition of glycerol (typically to a final concentration of 50%) for cryoprotection .

How can I design site-directed mutagenesis experiments for YnfA functional studies?

Based on previous mutagenesis studies of YnfA and knowledge from related SMR family transporters like EmrE, several approaches can be implemented:

• Identify conserved amino acid residues by sequence alignment with functionally characterized SMR family members.

• Target specific amino acids based on computational structural predictions.

• Focus on residues likely involved in substrate binding or transport mechanisms.

Previous studies have targeted several amino acid positions for mutation, including:

When designing mutagenesis experiments, use appropriate expression vectors (such as pBAD_Myc_HisA) and include controls with wild-type YnfA and empty vector .

What experimental approaches can be used to assess YnfA transporter activity?

YnfA transporter activity can be assessed using several complementary approaches:

• Resistance profiling using microtiter plate assays: Determine the minimum inhibitory concentration (MIC₉₀) of known SMR substrates such as ethidium bromide and acriflavine against strains expressing wild-type or mutant YnfA. The MIC₉₀ is defined as the lowest concentration at which 90% of bacterial growth is inhibited .

• Transport assays with fluorescent substrates: Measure the accumulation or efflux of fluorescent substrates (e.g., ethidium bromide) in cells expressing YnfA variants. This can be performed using fluorescence spectroscopy to monitor the kinetics of substrate transport.

• Competition assays: Assess the ability of unlabeled compounds to compete with fluorescent substrates, providing insights into substrate specificity.

• Growth inhibition assays: Compare growth curves of strains expressing different YnfA variants in the presence of varying concentrations of antimicrobial compounds.

When performing these assays, it's essential to include appropriate controls, such as cells harboring empty vectors, to accurately assess the contribution of YnfA to transport activity and resistance phenotypes .

How does YnfA contribute to antimicrobial resistance in bacteria?

YnfA functions as an efflux transporter belonging to the SMR family and plays a significant role in antimicrobial resistance. Studies have demonstrated that YnfA actively transports compounds like ethidium bromide and acriflavine out of the bacterial cell, reducing their intracellular concentration and thereby conferring resistance .

In Shigella flexneri, wild-type YnfA expression has been shown to provide a 2-fold greater resistance to ethidium bromide and a 4-fold greater resistance to acriflavine compared to control strains . This resistance profile is altered when key amino acid residues are mutated, particularly mutations at positions E15, G18, and Y60, which result in decreased resistance to these antimicrobial compounds .

The relevance of YnfA in clinical settings stems from its potential contribution to multidrug resistance in pathogenic bacteria, making it an important target for inhibitor development to combat antimicrobial resistance.

Which substrates are known to be transported by YnfA?

YnfA has been demonstrated to transport several substrates, primarily:

These compounds are well-established substrates for members of the SMR family of transporters. The substrate specificity profile of YnfA is likely broader, potentially including other cationic hydrophobic compounds, though comprehensive substrate profiling studies are still needed to fully characterize its transport capabilities .

How can computational approaches be used to predict YnfA structure and function?

Computational approaches provide valuable insights into YnfA structure and function:

• Homology modeling: Using the solved structure of related SMR family transporters like EmrE as templates, researchers can predict the three-dimensional structure of YnfA. This approach has been used to identify potential substrate binding sites and functional domains .

• Molecular dynamics simulations: These can model how YnfA interacts with the membrane environment and potential substrates, providing dynamic information about conformational changes during the transport cycle.

• Sequence alignment and conservation analysis: Identifying highly conserved residues across SMR family members can highlight functionally critical amino acids for experimental validation.

• Docking studies: In silico docking of potential substrates can predict binding modes and substrate specificity determinants.

These computational approaches are particularly valuable for membrane proteins like YnfA, where experimental structure determination can be challenging. Results from in silico studies can guide the design of targeted mutagenesis experiments to validate functional predictions .

What are the regulatory requirements for research involving recombinant YnfA?

Research involving recombinant YnfA should comply with institutional biosafety guidelines and, where applicable, NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. Based on the information provided:

• Experiments using well-established Escherichia coli K-12 host-vector systems may be exempt from the NIH Guidelines under certain conditions (as detailed in Appendix C of the NIH Guidelines) .

• While nucleic acid molecules resulting from the replication of recombinant DNA are subject to the NIH Guidelines, proteins produced by genetically engineered organisms (such as purified recombinant YnfA protein) are not subject to these guidelines .

• For experiments involving the cloning and expression of YnfA in E. coli, researchers should determine whether their specific host-vector system qualifies for exemption under Section III-F of the NIH Guidelines .

Researchers should consult with their Institutional Biosafety Committee (IBC) to ensure compliance with all relevant regulations and institutional policies before initiating research with recombinant YnfA .

How can I develop a high-throughput screening system for YnfA inhibitors?

Developing a high-throughput screening system for YnfA inhibitors could involve the following methodological approaches:

• Reporter-based systems: Engineer bacterial strains expressing YnfA along with a fluorescent or luminescent reporter system that responds to the intracellular accumulation of YnfA substrates. Compounds that inhibit YnfA would lead to increased substrate accumulation and a measurable signal change.

• Growth-based assays: Utilize the resistance phenotype conferred by YnfA by screening for compounds that sensitize YnfA-expressing bacteria to known substrates like ethidium bromide or acriflavine. This could be performed in a 96-well or 384-well format to enable high-throughput screening.

• Direct transport assays: Develop assays that directly measure the transport activity of YnfA using fluorescent substrates. Inhibitors would reduce the rate of substrate efflux from cells or membrane vesicles containing YnfA.

• Competitive binding assays: Use labeled ligands and purified YnfA protein to screen for compounds that compete for binding to the transporter.

For verification of hits, secondary assays should include dose-response studies, cytotoxicity assessment, and mechanism of action studies to confirm specific inhibition of YnfA rather than general membrane disruption or non-specific effects .

How should I interpret contradictory results in YnfA mutagenesis studies?

When encountering contradictory results in YnfA mutagenesis studies, consider the following methodological approach:

• Verify protein expression levels: Ensure that wild-type and mutant proteins are expressed at comparable levels using quantitative Western blotting. Differences in expression can confound functional comparisons .

• Check protein localization: Confirm that mutant proteins correctly localize to the membrane. Mutations may affect membrane integration, resulting in apparent loss of function.

• Consider substrate specificity shifts: Some mutations may alter substrate specificity rather than completely abolishing function. Test multiple substrates before concluding that a mutant is non-functional.

• Assess experimental conditions: Different assay conditions (pH, temperature, ionic strength) may differentially affect wild-type and mutant proteins.

• Examine potential structural implications: Using structural models, evaluate whether contradictory results might reflect complex structural interactions or compensatory mechanisms.

• Repeat experiments with additional controls: Include known functional and non-functional mutants as references for comparison.

In the case of YnfA, previous studies have shown that mutations like E15A, G18A, and Y60A significantly impact function, while other mutations (FF-LL, WLL-QVV, GGV-AAA, Y63A, Y67A, and Y86A) show no significant changes in resistance profile compared to wild-type YnfA . Contradictory results with these residues may warrant deeper investigation into specific experimental conditions or strain backgrounds.

What are common challenges in expressing and purifying functional recombinant YnfA?

Expressing and purifying functional membrane proteins like YnfA presents several challenges:

When working with YnfA, it's particularly important to ensure proper storage conditions. The lyophilized protein should be stored at -20°C/-80°C, with aliquoting recommended for multiple uses to avoid freeze-thaw cycles. For reconstituted protein, storage in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 is recommended .

How can I determine if mutations in YnfA affect protein stability versus transport function?

Distinguishing between effects on protein stability and transport function requires a multi-faceted approach:

• Protein expression and turnover analysis: Compare the steady-state levels and half-lives of wild-type and mutant YnfA proteins using pulse-chase experiments and Western blotting.

• Membrane localization studies: Use cell fractionation and/or fluorescent protein fusions to confirm proper membrane integration of mutant proteins.

• Thermal stability assays: If purification is feasible, compare the thermal denaturation profiles of wild-type and mutant proteins using techniques like differential scanning fluorimetry.

• Limited proteolysis: Assess structural integrity differences between wild-type and mutant proteins through their susceptibility to controlled proteolytic digestion.

• Substrate binding assays: Determine if mutants retain the ability to bind substrates even if transport function is compromised, which would suggest specific disruption of the transport mechanism rather than global protein destabilization.

• Complementation of multiple functional assays: Compare results from growth-based resistance assays with direct transport measurements. Discrepancies may indicate partial retention of function.

For YnfA specifically, mutations at positions E15, G18, and Y60 have been shown to significantly impact function, possibly affecting either substrate recognition, transport mechanism, or protein stability . Comprehensive analysis using the approaches above would help differentiate between these possibilities.

What novel approaches could advance our understanding of YnfA structure-function relationships?

Several cutting-edge approaches could significantly advance our understanding of YnfA:

• Cryo-electron microscopy (cryo-EM): This technique could potentially resolve the three-dimensional structure of YnfA at near-atomic resolution, providing crucial insights into its transport mechanism and substrate binding sites.

• Native mass spectrometry: This could reveal the oligomeric state of YnfA and identify potential interaction partners in the membrane.

• Single-molecule fluorescence resonance energy transfer (smFRET): This approach could capture conformational changes during the transport cycle, elucidating the dynamic aspects of YnfA function.

• Deep mutational scanning: Systematic mutation of each residue in YnfA followed by selection for function would generate a comprehensive map of functionally important residues.

• Ancestral sequence reconstruction: This evolutionary approach could provide insights into the functional diversification of SMR family transporters including YnfA.

• Synthetic biology approaches: Engineering synthetic circuits that couple YnfA activity to reporter outputs could enable new functional assays and selection strategies.

These approaches, combined with traditional biochemical and genetic methods, would provide a more comprehensive understanding of how YnfA structure relates to its function as an efflux transporter involved in antimicrobial resistance .

How might YnfA research contribute to addressing antimicrobial resistance?

YnfA research has significant potential to contribute to addressing antimicrobial resistance through multiple avenues:

• Inhibitor development: Detailed structural and functional characterization of YnfA could facilitate the design of specific inhibitors that block its efflux activity, potentially restoring the efficacy of existing antibiotics against resistant bacteria.

• Resistance mechanism elucidation: Understanding the substrate specificity and transport mechanism of YnfA contributes to our broader knowledge of how bacteria develop resistance through efflux mechanisms.

• Diagnostic applications: Knowledge of YnfA's role in resistance could lead to molecular diagnostic tools to identify resistant strains and guide treatment decisions.

• Combination therapy strategies: Identifying compounds that modulate YnfA activity might lead to novel combination therapies that enhance antibiotic efficacy.

• Evolutionary insights: Studying the conservation and diversity of YnfA across bacterial species could provide insights into the evolution of resistance mechanisms.

Previous research has demonstrated that YnfA contributes to resistance against compounds like ethidium bromide and acriflavine , suggesting its potential role in clinical antimicrobial resistance. Further research into its substrate specificity, particularly regarding clinically relevant antibiotics, would strengthen the connection between basic YnfA research and clinical applications in combating antimicrobial resistance.