Recombinant Escherichia coli UPF0060 membrane protein ynfA (ynfA)

How is recombinant YnfA typically expressed and purified?

Recombinant YnfA is commonly expressed in E. coli expression systems with affinity tags to facilitate purification. The most common approach involves:

• Expression as an N-terminal His-tagged fusion protein in E. coli

• Purification using affinity chromatography

• Storage as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE

For optimal protein stability, the purified protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with 5-50% glycerol added as a cryoprotectant for long-term storage at -20°C/-80°C . It's important to note that repeated freeze-thaw cycles should be avoided, and working aliquots can be stored at 4°C for up to one week .

What organisms express YnfA homologs and how evolutionarily conserved is this protein?

YnfA is relatively widespread among Gram-negative bacteria. Phylogenetic analysis has demonstrated that YnfA homologs can be found in various pathogenic Gram-negative bacteria with significant sequence conservation . Multiple sequence alignment and phylogenetic analysis have revealed that:

• The YnfA protein sequence of Shigella flexneri closely resembles the YnfA proteins found in E. coli, Salmonella, Citrobacter, Klebsiella, and Yersinia

• Most amino acids are highly conserved among YnfA homologs from different Gram-negative bacteria

• Phylogenetic analysis suggests that YnfA should be considered as a separate subfamily (YnfA family) within the SMR superfamily, alongside the three previously known subfamilies

This evolutionary conservation suggests important functional roles that have been maintained across bacterial species.

What methods have been used to determine or predict the 3D structure of YnfA?

The 3D structure of YnfA has been predicted using multiple computational approaches:

• I-TASSER (Iterative Threading ASSEmbly Refinement): This method utilized the already resolved crystal structure of the EmrE transporter (PDB ID: 3b61) from E. coli as a template to predict YnfA structure, with a coverage of 0.95 and a Normalized Z-score of 2.15, indicating good alignment and threading score

• AlphaFold protein structure database: This approach generated a 3D structure for YnfA that corroborated the I-TASSER results, confirming the four alpha-transmembrane helical model

The predicted structure closely resembles that of EmrE, another SMR family member, suggesting functional similarities. The structure consists of four alpha-transmembrane helices, which is consistent with the characteristic topology of SMR family transporters .

What role does YnfA play in antimicrobial resistance?

YnfA functions as an efflux transporter that contributes to antimicrobial resistance in bacterial pathogens. Studies in Shigella flexneri have demonstrated that YnfA promotes resistance to various antimicrobial compounds by actively exporting these substances from the bacterial cell .

Research has identified YnfA as a potential target for developing inhibitors that could be used to combat antimicrobial resistance. By disrupting the function of YnfA and similar efflux pumps, it may be possible to increase the susceptibility of resistant bacterial strains to existing antibiotics . Experimental approaches to assess YnfA's role in resistance have included:

• Gene knockout studies to assess changes in minimum inhibitory concentrations (MICs)

• Transport assays to measure efflux activity

• Assessment of the effects of disrupting either one or two SMR efflux pumps on the resistance profile of Shigella flexneri

These methodologies provide a framework for researchers investigating the contribution of YnfA to antimicrobial resistance in various bacterial species.

How does expression of recombinant YnfA compare to other membrane proteins?

Membrane proteins like YnfA present significant challenges for recombinant expression due to their hydrophobic surfaces. Recent advances have developed approaches to overcome these challenges:

• Water-soluble RFdiffused Amphipathic Proteins (WRAPs): A deep learning-based design approach that creates proteins to surround the lipid-interacting hydrophobic surfaces of membrane proteins, rendering them stable and water-soluble without the need for detergents . This approach preserves the sequence, fold, and function of the native membrane protein .

• Specialized expression vectors: Systems like pGRASS (Green fluorescent protein Reporter from Antisense promoter-based Screening System) have been engineered for improved cloning and expression of recombinant proteins in E. coli, offering increased plasmid copy number (approximately 3-fold higher than wild-type pET3b vector) and enhanced fluorescence for easier screening .

When expressing YnfA, researchers should consider:

• Using weak constitutive promoters rather than strong inducible promoters to minimize toxicity

• Optimizing codon usage to match the expression host

• Employing fusion tags that enhance solubility or membrane integration

What site-directed mutagenesis approaches have been used to identify critical residues in YnfA?

Mutagenesis studies of YnfA have built upon knowledge gained from related transporters like EmrE to identify crucial amino acid residues . The approach typically includes:

• Selecting target amino acids based on sequence conservation and structural predictions

• Creating point mutations using site-directed mutagenesis

• Assessing the impact of mutations on protein function through transport assays and resistance profiles

Key findings from these studies suggest that conserved residues in the three motif blocks identified through multiple sequence alignment are likely critical for YnfA function . Researchers investigating YnfA should focus on these conserved regions when designing mutagenesis experiments to further characterize structure-function relationships.

How can codon optimization improve recombinant YnfA expression?

Codon optimization can significantly impact the expression level of recombinant proteins like YnfA in E. coli. Analysis of relative codon bias (RCB) can help predict gene expression levels and guide optimization strategies .

When expressing YnfA in E. coli, researchers should consider:

• Expression environment effects: Gene expression levels can vary significantly between different growth conditions. For example, correlation coefficients between expression levels from different experiments (rLP-SP = 0.52, rLB-LP = 0.017, rLB-SP = -0.039) show substantial variation, highlighting the importance of optimizing expression conditions specifically for YnfA .

• Multifactorial optimization: While codon usage is important, other factors such as promoter strength and gene copy number should also be considered for optimal expression . The pGRASS vector system, with its increased copy number due to a single nucleotide deletion in the origin of replication, demonstrates how such factors can enhance expression levels .

• Culture media selection: E. coli cultured in different media (such as lysogeny broth) can show varying expression patterns that might affect YnfA production .

What solubilization strategies are most effective for membrane proteins like YnfA?

Solubilizing membrane proteins like YnfA while maintaining their native structure and function presents a significant challenge. Several approaches can be considered:

• Traditional detergent solubilization: Using mild detergents like n-dodecyl-β-D-maltoside (DDM) or n-octyl-β-D-glucopyranoside (OG)

• Designed protein WRAPs: A recently developed approach using Water-soluble RFdiffused Amphipathic Proteins that surround hydrophobic surfaces of membrane proteins, rendering them stable and water-soluble without detergents . This method has shown success with both beta-barrel outer membrane and helical multi-pass transmembrane proteins .

• Reconstitution buffers: For YnfA specifically, reconstitution in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 has been reported to be effective .

When working with YnfA, it's important to consider that solubilization conditions must be optimized to maintain the four-transmembrane helical structure that is critical for function.

How can researchers assess the functional activity of recombinant YnfA?

To evaluate whether recombinant YnfA maintains its native functional activity, several experimental approaches can be employed:

• Transport assays: Measuring the efflux of known substrates using fluorescent dyes or radiolabeled compounds

• Antimicrobial susceptibility testing: Comparing minimum inhibitory concentrations (MICs) in strains expressing wild-type YnfA versus mutant variants or knockouts

• Binding studies: Assessing the interaction between YnfA and potential inhibitors or substrates using techniques such as isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR)

• Electrophysiology: Reconstituting YnfA in lipid bilayers to measure transport activity at the single-protein level

These approaches can be combined to provide a comprehensive characterization of YnfA function, particularly its role in antimicrobial resistance.

What are the most promising applications for further YnfA research?

Based on current understanding of YnfA, several promising research directions emerge:

• Antimicrobial development: As YnfA contributes to antimicrobial resistance, developing specific inhibitors could help combat drug-resistant bacterial infections

• Structural biology: Further refinement of YnfA's structural model could provide insights into the mechanisms of substrate recognition and transport

• Evolutionary studies: Comparative analysis of YnfA homologs across different bacterial species could reveal evolutionary adaptations and functional specializations

• Synthetic biology applications: Engineered YnfA variants might be developed for biotechnological applications, such as biosensors or selective transport systems