Recombinant Staphylococcus aureus UPF0754 membrane protein SAHV_1831 (SAHV_1831)

What is Staphylococcus aureus UPF0754 membrane protein SAHV_1831?

Staphylococcus aureus UPF0754 membrane protein SAHV_1831 (Uniprot: A7X3V5) is a membrane-spanning protein from Staphylococcus aureus strain Mu3/ATCC 700698 . The protein consists of 374 amino acids with multiple transmembrane domains and belongs to the UPF0754 protein family . As a membrane-embedded protein, SAHV_1831 contains hydrophobic regions that anchor it within the bacterial cell membrane, suggesting its potential role in bacterial membrane integrity, transport, or signaling pathways . The protein's full amino acid sequence begins with MNALFIIFMIVVGAIIGGITNVIAI and continues through multiple hydrophobic and hydrophilic regions, characteristic of integral membrane proteins that traverse the lipid bilayer .

What expression systems are optimal for producing recombinant SAHV_1831?

The optimal expression system for recombinant SAHV_1831 production depends on research objectives and downstream applications. For high-yield production, Escherichia coli-based expression systems have demonstrated efficacy for membrane proteins similar to SAHV_1831, allowing for cytoplasmic membrane localization with the intended transmembrane topology . Successful expression protocols typically utilize E. coli strains optimized for membrane protein expression (such as C41/C43 or Lemo21) combined with inducible promoters that allow careful control of expression rates . Alternative expression hosts include yeast systems, which have shown success with other Staphylococcus aureus proteins and can provide eukaryotic post-translational modifications if required . Expression optimization should address inducer concentration, temperature, and duration parameters through systematic testing, as membrane protein overexpression can lead to toxicity, misfolding, or inclusion body formation that reduces functional yield .

What purification strategies yield the highest purity and activity of SAHV_1831?

Purification of SAHV_1831 requires specialized approaches to maintain protein integrity while removing the lipid bilayer. The initial extraction step should utilize mild detergents that effectively solubilize the membrane without denaturing the protein structure . Detergents such as n-dodecyl-β-D-maltoside (DDM), lauryl maltose neopentyl glycol (LMNG), or digitonin have proven effective for membrane protein extraction while preserving structural integrity . Following extraction, affinity chromatography utilizing engineered tags (typically His-tags) enables specific capture of the target protein . This initial purification should be followed by size exclusion chromatography to separate protein-detergent complexes from aggregates and contaminants . Throughout purification, protein stability should be monitored using techniques such as circular dichroism or fluorescence spectroscopy to ensure the helical secondary structure consistent with the original design is maintained . For applications requiring higher purity (>90%), additional ion exchange or hydrophobic interaction chromatography steps may be incorporated into the purification workflow .

How can researchers effectively study the membrane topology of SAHV_1831?

Determining the membrane topology of SAHV_1831 requires complementary experimental approaches to map the orientation and organization of transmembrane domains. Computational prediction tools like TMHMM, MEMSAT, and PredictProtein provide initial models of transmembrane regions, but experimental validation is essential . Cysteine scanning mutagenesis, where single cysteine residues are introduced throughout the protein sequence followed by accessibility testing with membrane-impermeable sulfhydryl reagents, can map exposed versus embedded regions . Protease protection assays, which exploit differential protease accessibility to cytoplasmic versus periplasmic protein domains, provide additional topological information when performed on membrane vesicles . Fluorescence-based approaches using environment-sensitive probes attached to specific residues can detect membrane insertion depth and local environment polarity . For higher resolution analysis, hydrogen-deuterium exchange mass spectrometry (HDX-MS) reveals solvent-accessible regions versus membrane-protected segments, providing dynamic structural information that complements static methods . These combined approaches generate comprehensive topological maps that inform structure-function relationships and guide rational engineering efforts.

What are the most effective methods for determining the three-dimensional structure of SAHV_1831?

Determining the three-dimensional structure of SAHV_1831 presents significant challenges due to its membrane-embedded nature. X-ray crystallography remains a powerful approach but requires the production of well-diffracting crystals, which can be facilitated using antibody Fab fragments as crystallization chaperones to stabilize specific conformations . These chaperones can be generated through phage display selection targeting SAHV_1831 embedded in lipid nanodiscs to maintain native-like conformations during the selection process . Single-particle cryo-electron microscopy (cryo-EM) offers an alternative approach that avoids crystallization requirements, particularly effective for larger membrane proteins or complexes where Fab fragments can serve as fiducial markers to aid particle alignment and three-dimensional reconstruction . Nuclear magnetic resonance (NMR) spectroscopy, particularly solution NMR of detergent-solubilized protein or solid-state NMR of reconstituted protein in lipid bilayers, provides complementary structural and dynamic information . For each methodology, stabilization of SAHV_1831 in a native-like lipid environment is critical, achievable through detergent micelles carefully selected to mimic native membrane environments or through incorporation into nanodiscs or lipid cubic phase systems .

How can researchers capture and stabilize different conformational states of SAHV_1831 for structural studies?

Capturing transient conformational states of SAHV_1831 presents a significant technical challenge, as these states are often too fleeting for conventional structural analysis techniques . A powerful approach combines antibody Fab-based phage display selection with nanodisc technology to generate conformation-selective binding proteins . This methodology involves embedding SAHV_1831 in lipid-filled nanodiscs that provide a native-like membrane environment while exposing epitopes accessible for antibody binding . The selection conditions can be manipulated (through ligand addition, buffer composition, or temperature variation) to enrich for Fabs that preferentially bind to and stabilize specific conformational states . The resulting conformation-selective antibody fragments can effectively "lock" the protein in a desired conformational form, allowing subsequent structural characterization . Alternative approaches include designing disulfide bridges between strategically positioned cysteine residues to constrain the protein in specific conformations or utilizing mutations in hinge regions that alter conformational equilibria . Small molecule stabilizers identified through computational docking or high-throughput screening can also prove valuable for trapping functionally relevant states of membrane proteins .

What approaches can be used to determine the biological function of SAHV_1831?

Determining the biological function of SAHV_1831 requires a multi-faceted approach combining genetic, biochemical, and biophysical methodologies. Gene knockout or silencing experiments in S. aureus, followed by phenotypic characterization, can reveal physiological roles through growth defects, altered membrane properties, or stress response changes . Protein-protein interaction studies using techniques such as bacterial two-hybrid systems, co-immunoprecipitation, or cross-linking mass spectrometry can identify binding partners that suggest functional pathways . Additionally, reconstitution of purified SAHV_1831 into proteoliposomes or planar lipid bilayers enables functional assays for potential transport or channel activities through measurement of ion flux, substrate translocation, or membrane potential changes . Binding assays with potential ligands or cofactors, similar to the heme coordination observed in engineered membrane proteins, can reveal interaction partners and suggest enzymatic functions . Computational approaches including structural homology modeling and phylogenetic analysis provide complementary insights by identifying conserved domains and evolutionary relationships that may suggest functional roles . Integration of these diverse data sources through systems biology approaches can ultimately position SAHV_1831 within specific cellular pathways and processes.

What methods are most effective for studying protein-lipid interactions of SAHV_1831?

Understanding protein-lipid interactions is crucial for comprehending SAHV_1831 function, as these interactions can modulate protein structure, orientation, dynamics, and activity. Reconstituting purified SAHV_1831 into liposomes of defined lipid composition allows systematic investigation of how different lipids affect protein stability and function . Fluorescence techniques using environment-sensitive probes attached to strategic positions can detect conformational changes induced by specific lipids or membrane properties . Hydrogen-deuterium exchange mass spectrometry reveals lipid-protected regions of the protein, identifying potential lipid binding sites or membrane-embedded segments . Molecular dynamics simulations provide atomistic insights into protein-lipid interactions, revealing preferential associations, lipid sorting effects, and bilayer deformations induced by the protein . Native mass spectrometry techniques adapted for membrane proteins can directly detect specifically bound lipids that remain associated with the protein during analysis, distinguishing specific binding from bulk solvation effects . Nanodiscs with controlled lipid composition offer particular advantages, as they provide a native-like membrane environment while allowing precise control over lipid species, facilitating correlation between specific lipids and conformational or functional properties .

How can SAHV_1831 be utilized as a model system for membrane protein design and engineering?

SAHV_1831 offers valuable opportunities as a model system for membrane protein design principles, particularly for investigating sequence-structure-function relationships in multi-spanning membrane proteins. Researchers can exploit SAHV_1831's multi-helical architecture to explore minimal amino acid requirements for membrane protein folding and stability, similar to studies with designer membrane proteins that demonstrated functional properties using only four amino acids (L, S, G, and W) in transmembrane domains . Systematic substitution of transmembrane residues can reveal essential packing interactions, while modifications to connecting loops can examine flexibility requirements for proper folding and conformational dynamics . The introduction of functional motifs such as ion coordination sites, substrate binding pockets, or dimerization interfaces enables investigation of how structural elements contribute to specificity and activity . Comparative studies between SAHV_1831 and other UPF0754 family members can identify conserved structural principles versus variable regions that confer unique properties . Additionally, the protein can serve as a scaffold for developing novel biosensors, where engineered binding sites or conformational changes translate molecular recognition events into detectable signals . These design principles generate fundamental insights applicable to synthetic biology and therapeutic development targeting membrane proteins.

What are the challenges and solutions for incorporating SAHV_1831 into nanodiscs for structural and functional studies?

Incorporating SAHV_1831 into nanodiscs presents specific challenges that require methodological refinement for successful implementation. The primary challenge involves optimizing reconstitution conditions to ensure proper protein insertion and orientation within the nanodisc lipid bilayer . Successful protocols typically utilize a detergent-mediated reconstitution approach, where detergent-solubilized SAHV_1831 is combined with lipids and membrane scaffold proteins (MSPs), followed by controlled detergent removal through dialysis or adsorption to bio-beads . Lipid composition critically impacts reconstitution efficiency and protein function, requiring systematic testing of various phospholipid mixtures that mimic bacterial membranes (typically phosphatidylglycerol and cardiolipin mixtures) . The MSP:lipid:protein ratio must be optimized to prevent protein aggregation while ensuring monodisperse nanodisc formation, typically assessed through size exclusion chromatography and negative-stain electron microscopy . Functional verification using activity assays is essential to confirm that nanodisc-embedded SAHV_1831 retains native-like behavior compared to detergent-solubilized or native membrane forms . Advanced applications include generating SAHV_1831-loaded nanodiscs of controlled size and stoichiometry by selecting appropriate MSP variants, enabling specific experimental applications from single-particle cryo-EM to surface-based sensor technologies .

How can phage display technology be leveraged to generate conformation-specific antibodies against SAHV_1831?

Generating conformation-specific antibodies against SAHV_1831 through phage display technology requires specialized approaches to maintain the protein's native conformation during selection. The process begins with preparing SAHV_1831 in multiple conformational states, achieved through manipulation of buffer conditions, addition of putative ligands, or introduction of mutations that shift conformational equilibria . Embedding SAHV_1831 in lipid nanodiscs provides significant advantages over detergent-solubilized formats by maintaining a native-like lipid environment that preserves conformational integrity throughout the selection process . The selection strategy should employ subtractive approaches, where phage libraries are first depleted of binders to undesired conformations before positive selection against the target conformation . Synthetic antibody libraries (sABs) with diversified complementarity-determining regions provide a rich source of potential binders without requiring animal immunization . Multiple rounds of selection with increasing stringency progressively enrich for high-affinity, conformation-specific binders . Validation of selected antibody fragments should combine binding assays (ELISA, surface plasmon resonance) with functional assays to confirm their conformational specificity and ability to stabilize particular states . The resulting conformation-selective antibodies serve as valuable tools for structural studies, enabling crystallization of specific conformations, providing fiducial markers for cryo-EM, and stabilizing transient states for biophysical characterization .

How can researchers overcome issues with SAHV_1831 expression and aggregation?

Membrane protein expression and aggregation challenges require systematic optimization strategies to obtain properly folded SAHV_1831. Low expression yields can be addressed by modifying the expression construct to incorporate fusion partners that enhance folding and membrane targeting, such as Mistic, GFP, or MBP tags positioned at the N- or C-terminus . Expression conditions should be optimized by reducing induction temperature (typically to 16-20°C) and lowering inducer concentration to slow production rates, allowing proper membrane insertion machinery engagement . Specialized E. coli strains like C41(DE3), C43(DE3), or Lemo21(DE3) designed specifically for membrane protein expression can significantly improve yields by mitigating toxicity issues . For proteins prone to aggregation during extraction, screening multiple detergents is crucial, beginning with mild options like DDM, LMNG, or GDN before testing harsher alternatives if necessary . Addition of stabilizing agents such as glycerol, specific lipids, or putative ligands during extraction can preserve native conformations and prevent aggregation . Protein engineering approaches including surface residue substitutions to increase solubility or removal of flexible regions prone to aggregation may be necessary for particularly challenging constructs . If conventional approaches fail, cell-free expression systems offer an alternative that allows direct incorporation into liposomes or nanodiscs during synthesis, bypassing extraction challenges entirely .

What strategies can resolve difficulties in obtaining high-resolution structural data for SAHV_1831?

Obtaining high-resolution structural data for membrane proteins like SAHV_1831 presents significant challenges that require specialized approaches. For X-ray crystallography, crystal quality can be improved by systematically screening constructs with modified termini, loop regions, or surface residues that may interfere with crystal contacts . Lipidic cubic phase (LCP) crystallization methods often yield better-diffracting crystals for membrane proteins compared to vapor diffusion approaches by providing a more native-like environment during crystal formation . For cryo-EM studies, protein stability in ice can be enhanced through careful grid preparation protocols, including the use of graphene oxide or specialized grid surfaces that prevent protein denaturation at the air-water interface . The use of antibody fragments (Fabs) selected through phage display offers dual benefits: enhancing protein stability and providing additional mass to facilitate particle alignment during image processing . When traditional approaches yield limited resolution, integrative structural biology combining lower-resolution data (small-angle X-ray scattering, cryo-EM) with computational modeling and molecular dynamics simulations can generate meaningful structural models . For particularly challenging targets, cross-linking mass spectrometry provides distance constraints that inform computational models even in the absence of high-resolution structures . Multiple structural biology approaches should be pursued in parallel to maximize the chances of success while providing complementary structural information.

How can researchers validate that recombinant SAHV_1831 maintains native-like structure and function?

Validating native-like structure and function of recombinant SAHV_1831 requires multiple complementary approaches. Circular dichroism spectroscopy provides essential information on secondary structure content, confirming the expected alpha-helical characteristics of properly folded membrane proteins . Thermal or chemical denaturation studies monitored by spectroscopic methods reveal stability profiles that can be compared between different preparation methods or with predicted values . Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) assesses protein monodispersity and oligomeric state, crucial indicators of proper folding . Lipid binding assays using native lipid extracts from S. aureus can evaluate whether the recombinant protein properly interacts with its natural lipid environment . Where possible, specific functional assays should be developed based on predicted activities (transport, signaling, or enzymatic functions) and compared with native membrane preparations . For proteins without known functions, structural integrity can be indirectly assessed through conformation-specific antibody binding or ligand interaction studies . Computational approaches including molecular dynamics simulations of the recombinant protein in membrane environments provide additional validation by assessing structural stability and dynamics over extended timescales . Integration of these diverse validation methods builds confidence that the recombinant protein maintains biologically relevant structural and functional properties.