Recombinant Escherichia coli O127:H6 UPF0060 membrane protein ynfA (ynfA)

What is YnfA and what structural family does it belong to?

YnfA is a small membrane protein (11.9 kDa) comprising 108 amino acids and functions as an efflux transporter in Escherichia coli. It belongs to the Small Multidrug Resistance (SMR) superfamily of transporters. Structural prediction analyses using tools like TMHMM and TMpred reveal that YnfA consists of four α-transmembrane helices, which is a characteristic signature of members in the SMR superfamily. The protein is encoded at gene position 1616397 to 1616723 in the E. coli genome and functions as an integral inner membrane protein .

How is YnfA phylogenetically related to other membrane transporters?

Phylogenetic analysis using the MEGA software and maximum composite likelihood method demonstrates that YnfA is a distant homolog of other SMR family members. Based on multiple sequence alignments, YnfA should be considered as a separate subfamily (YnfA family) alongside the three previously known subfamilies of SMR transporters. This phylogenetic distinction suggests unique structural or functional properties that differentiate YnfA from other membrane transporters. The protein's homologs have been identified in various Gram-negative pathogenic bacteria, indicating its widespread occurrence and potential evolutionary significance .

What are the key challenges in purifying YnfA and other membrane proteins?

Purification of YnfA presents significant challenges due to its hydrophobic nature. Like other membrane proteins, YnfA tends to aggregate and precipitate in aqueous solutions, requiring incorporation into detergents or lipids that mimic its native environment. The purification process must maintain protein stability while removing cellular contaminants. Even in the presence of detergents or lipids, membrane proteins like YnfA may exhibit aggregation tendencies that complicate purification efforts. Researchers typically need to optimize detergent types, concentrations, and buffer compositions to successfully isolate milligram quantities of functional YnfA for structural and functional studies .

What storage conditions maximize YnfA stability?

For optimal stability, purified YnfA should be stored at -20°C or -80°C for extended storage. The recommended storage buffer consists of a Tris-based buffer with 50% glycerol, which has been optimized for this protein. Working aliquots can be maintained at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided as they can compromise protein integrity. For reconstitution, it is advised to use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% is recommended for long-term storage, with 50% being the standard concentration used in commercial preparations .

What hybrid methods can effectively study YnfA in native membranes?

A powerful approach for studying YnfA in its native membrane environment combines high-resolution solid-state NMR spectroscopy (ssNMR) with electron cryotomography (cryoET). This hybrid methodology allows researchers to maintain the protein in its natural lipid bilayer context without using detergents or extraction procedures that might alter its structure or function. The combined approach leverages the complementary strengths of each technique: ssNMR provides atomic-level structural and dynamical information, while cryoET offers environmental context and larger structural details through subvolume averaging. The experimental protocol typically involves generating isotopically labeled, rifampicin-treated bacteria that are gently disrupted to produce cell envelope particles suitable for both analytical methods .

How does solid-state NMR complement other structural analysis techniques for YnfA?

Solid-state NMR (ssNMR) provides unique advantages when studying membrane proteins like YnfA, especially when complementing other structural techniques. Unlike X-ray crystallography, ssNMR doesn't require protein crystallization, which is particularly challenging for membrane proteins. ssNMR excels at providing Ångstrom-scale chemical information and capturing dynamic properties that are implicitly included in the measurements. The technique can exploit isotope labeling to exclude background signals, enabling focused analysis of the protein of interest. When combined with techniques like cryoET, which provides nanometer-scale spatial information and environmental context, researchers can develop comprehensive structural models of YnfA in its native membrane environment. This complementary approach bridges the gap between atomic-level details and larger contextual information necessary for understanding membrane protein function .

What computational approaches aid in predicting YnfA structure?

Computational prediction of YnfA structure has been successfully achieved using methods like I-TASSER, which can use structural homologs like EmrE as templates. These predictions have revealed that YnfA's structure closely resembles that of EmrE, with coverage of 0.95 and a Normalized Z-score of 2.15, indicating good alignment and threading score. The AlphaFold protein structure database also corroborates the four alpha-transmembrane helical model predicted by I-TASSER. These computational approaches provide valuable structural insights, especially when experimental structure determination is challenging. The predicted structures can guide experimental design and interpretation, helping researchers understand potential functional mechanisms based on structural features .

What functional role does YnfA play as an efflux transporter?

YnfA functions as an efflux transporter involved in resistance mechanisms in E. coli. As a member of the SMR superfamily, it likely contributes to the extrusion of toxic compounds from the bacterial cell, potentially including antibiotics and other antimicrobial agents. The protein contains three conserved motif blocks that are considered indispensable for appropriate functioning of SMR transporters. These motifs likely play crucial roles in substrate recognition, binding, and transport mechanisms across the membrane. Understanding YnfA's role in efflux transport provides insights into bacterial resistance mechanisms and potential targets for antimicrobial development .

How do the conserved motifs in YnfA contribute to its function?

Multiple sequence alignment of YnfA with other SMR family transporters has revealed three conserved motif blocks that are essential for proper functioning. These conserved regions likely play critical roles in substrate binding, conformational changes during transport, and energy coupling. The specific arrangement of these motifs within the four transmembrane helices creates the structural framework necessary for transporter activity. Mutation studies of these conserved regions would provide valuable insights into structure-function relationships, potentially identifying key residues involved in substrate specificity or transport efficiency. The conservation of these motifs across SMR family members suggests fundamental mechanistic similarities despite the evolutionary divergence of YnfA as a distinct subfamily .

What experimental approaches can assess YnfA transport activity?

Assessment of YnfA transport activity can employ several experimental approaches. Reconstitution of purified YnfA into liposomes or nanodiscs containing appropriate fluorescent dyes can enable real-time monitoring of transport activity. Substrate accumulation or efflux assays using radiolabeled or fluorescent compounds can quantify transport kinetics. Electrophysiological techniques like patch-clamp or black lipid membrane studies can measure ion currents associated with transport. Additionally, growth inhibition assays with various potential toxic substrates, comparing wild-type and YnfA-knockout strains, can identify physiologically relevant substrates. These approaches can be complemented by binding studies using isothermal titration calorimetry or surface plasmon resonance to characterize substrate interactions .

How can lipid nanodiscs be utilized for studying YnfA structure and function?

Lipid nanodiscs provide an ideal membrane-mimetic environment for studying YnfA structure and function. These disc-shaped lipid bilayer patches, encircled by membrane scaffold proteins, offer several advantages: they maintain the protein in a native-like lipid environment, provide stability in aqueous solutions, and are compatible with various biophysical techniques. For YnfA studies, researchers can reconstitute the purified protein into nanodiscs composed of appropriate phospholipids that mimic the E. coli inner membrane. These YnfA-containing nanodiscs can then be analyzed by solution NMR, solid-state NMR, cryo-EM, or functional assays to correlate structural features with transport activity. The nanodisc system allows controlled manipulation of the lipid environment, enabling studies on how membrane composition affects YnfA structure and function .

What strategies can overcome expression challenges for toxic membrane proteins like YnfA?

Expression of potentially toxic membrane proteins like YnfA requires specialized strategies to minimize cellular stress while maximizing protein yield. Researchers can employ tightly regulated expression systems with inducible promoters that allow precise control over expression timing and level. Using specialized E. coli strains designed for toxic protein expression (such as C41/C43 or LEMO21) can improve tolerance to membrane protein overexpression. Fusion partners that enhance folding or reduce toxicity (such as Mistic, SUMO, or GFP) can be employed. Cold induction protocols (expression at 16-20°C) often reduce toxicity while improving proper folding. Additionally, co-expression with chaperones may enhance proper membrane insertion. Finally, optimizing media composition and induction parameters through Design of Experiments (DoE) approaches can identify optimal conditions that balance protein yield with cell viability .

How can mutagenesis studies advance our understanding of YnfA function?

Systematic mutagenesis studies of YnfA can provide critical insights into structure-function relationships. Site-directed mutagenesis targeting conserved residues identified through sequence alignments can reveal amino acids essential for substrate binding, transport mechanism, or protein stability. Alanine-scanning mutagenesis across transmembrane segments can identify functional hotspots. Creating chimeric proteins by swapping domains between YnfA and related transporters can help define regions responsible for substrate specificity. Introducing reporter residues (e.g., cysteine mutants for accessibility studies or fluorescent probes) can enable dynamic conformational analysis during transport cycles. Combining these mutagenesis approaches with functional assays and structural studies provides a powerful framework for dissecting the molecular mechanisms of YnfA-mediated transport and potentially identifying residues that could be targeted for inhibitor development .

What are the most effective membrane mimetics for functional studies of YnfA?

Selecting appropriate membrane mimetics is crucial for maintaining YnfA's native structure and function during in vitro studies. While conventional detergents like DDM, DM, or LDAO can solubilize YnfA, they may not fully preserve its functional state. Lipid nanodiscs and macrodiscs provide superior environments by maintaining the protein within a lipid bilayer. These disc systems allow for controlled lipid composition that can mimic the E. coli inner membrane, potentially using E. coli polar lipid extracts or defined mixtures of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Other effective mimetics include bicelles (lipid-detergent mixtures), amphipols (amphipathic polymers), and styrene-maleic acid lipid particles (SMALPs) that can extract membrane proteins with their surrounding lipids directly from membranes. The choice between these systems depends on the specific experimental technique and research question, with consideration for stability, homogeneity, and compatibility with analytical methods .

What challenges exist in resolving YnfA structure at atomic resolution?

Resolving the atomic structure of YnfA faces several significant challenges inherent to membrane protein structural biology. Obtaining sufficient quantities of pure, stable, and homogeneous protein samples remains difficult despite advanced expression and purification protocols. Crystallization for X-ray crystallography is particularly challenging due to YnfA's small size and hydrophobic nature, which complicates crystal formation with ordered packing. For cryo-EM studies, the small size of YnfA (11.9 kDa) falls below the typical detection limit, though advances in microscopy technology are gradually lowering this threshold. NMR studies require isotopic labeling and optimization of conditions that maintain native-like conformations while providing sufficient spectral resolution. Additionally, capturing different conformational states relevant to the transport cycle presents another layer of complexity. Overcoming these challenges requires integrated approaches combining multiple complementary techniques and potentially novel methodologies specifically adapted for small membrane proteins .