Recombinant Helicobacter pylori Membrane protein insertase YidC (yidC)
Description
Introduction to Membrane Protein Insertase YidC
YidC belongs to a conserved family of membrane protein insertases found across bacterial species, with homologs in mitochondria (Oxa1) and chloroplasts (Alb3) of eukaryotes. In bacterial systems such as Escherichia coli, YidC plays a crucial role in facilitating the insertion of proteins into the cytoplasmic membrane, which is essential for proper cellular function and survival . This protein operates through two primary mechanisms: in cooperation with the Sec translocon or independently as a dedicated insertase for specific substrate proteins.
Beyond its insertase function, YidC serves as a foldase that promotes proper assembly of membrane protein complexes. Known YidC-only substrates identified in E. coli studies include the F₁F₀-ATPase subunit c, M13 phage procoat protein, mechano-sensing MscL protein, Sci-1 type VI secretion system subunit TssL, and Pf3 coat protein . This dual functionality as both insertase and foldase underscores YidC's importance in membrane protein biogenesis pathways across bacterial species.
Functional Mechanism in Membrane Protein Biogenesis
The functional mechanism of YidC in Helicobacter pylori can be inferred from extensive studies conducted in model bacterial systems, particularly E. coli. YidC facilitates the insertion of membrane proteins through two principal pathways that likely operate similarly in H. pylori :
The first pathway involves Sec-dependent insertion, where YidC works cooperatively with the Sec translocon. In this process, nascent membrane proteins initially engage with the Sec machinery, which facilitates their translocation across or insertion into the membrane. YidC then receives these proteins from the Sec translocon, assisting in their proper folding, assembly, and final positioning within the lipid bilayer .
The second pathway involves Sec-independent insertion, where YidC functions autonomously to insert specific substrates directly into the membrane. These "YidC-only" substrates typically possess short periplasmic domains and specific hydrophobic characteristics that allow them to bypass the Sec machinery. This independent insertase activity highlights YidC's versatility in membrane protein biogenesis .
In both pathways, YidC likely provides a protected environment for hydrophobic domains of substrate proteins, preventing misfolding and aggregation during the insertion process. The protein's ability to facilitate the proper assembly of membrane protein complexes further emphasizes its critical role in maintaining cellular function and integrity.
Recombinant Expression Systems and Purification
The recombinant expression of H. pylori proteins has been successfully demonstrated for several proteins, including catalase . Similar methodologies can be applied to express recombinant H. pylori YidC for structural and functional studies. Based on established protocols for other H. pylori proteins and related YidC proteins, the expression of recombinant H. pylori YidC would involve several key steps.
First, the yidC gene must be amplified from H. pylori chromosomal DNA using PCR techniques with specific primers designed based on the known sequence of the gene . This amplified gene is then inserted into an appropriate expression vector, commonly pET-series vectors that are widely used for recombinant protein expression in E. coli . The recombinant vector is subsequently transformed into an expression host, typically E. coli strains like BL21(DE3), followed by induction of protein expression using IPTG (Isopropyl β-D-1-thiogalactopyranoside) .
Following expression, the recombinant protein can be purified using affinity chromatography, facilitated by the addition of affinity tags such as histidine tags. The recombinant H. hepaticus YidC has been successfully expressed with an N-terminal His-tag in E. coli, resulting in high purity protein suitable for downstream applications . Similar approaches would likely prove effective for H. pylori YidC.
Table 2: Recommended Conditions for Recombinant H. pylori YidC Expression
Once purified, the recombinant protein should be stored appropriately to maintain its stability and activity. For related YidC proteins, storage in a Tris/PBS-based buffer with trehalose at -20°C/-80°C with the addition of glycerol has proven effective .
Applications in Research and Therapeutic Development
Recombinant H. pylori YidC has numerous potential applications spanning basic research and applied biomedical fields. In structural biology, purified recombinant YidC can be utilized for crystallography or cryo-electron microscopy to determine its three-dimensional structure, providing critical insights into its mechanism of action. Such structural information would enhance our understanding of membrane protein biogenesis in this significant human pathogen.
Functional characterization represents another important application, where in vitro assays with recombinant YidC can help identify specific substrates in H. pylori and elucidate its role in membrane protein assembly. Understanding which proteins depend on YidC for proper insertion could reveal crucial aspects of H. pylori cellular physiology and pathogenesis.
From a therapeutic perspective, YidC represents a potential target for novel antimicrobial development against H. pylori. Given the essential role of YidC in bacterial viability and its conservation across bacterial species, inhibitors targeting this protein could potentially provide new approaches to combat H. pylori infections . This is particularly relevant given the increasing prevalence of antibiotic resistance in clinical H. pylori isolates, which has complicated treatment strategies.
Additionally, recombinant H. pylori YidC could facilitate pathogenesis studies by helping researchers understand how this protein contributes to the insertion of virulence factors and other proteins important for colonization and survival in the harsh gastric environment. Such insights could potentially identify new strategies for preventing or treating H. pylori-associated diseases, including gastritis, peptic ulcers, and gastric cancer .
Current Research Challenges and Future Directions
Despite its biological significance, research specifically focused on H. pylori YidC faces several challenges. The expression and purification of membrane proteins like YidC presents technical difficulties due to their hydrophobic nature and tendency to aggregate. Additionally, determining the specific substrates of H. pylori YidC requires specialized techniques that may not be widely available.
Future research directions should focus on comprehensive characterization of H. pylori YidC structure using advanced techniques such as cryo-electron microscopy or X-ray crystallography. Identification of its specific substrates through proteomic approaches would provide valuable insights into its role in H. pylori physiology and pathogenesis. Furthermore, exploring YidC's potential as a therapeutic target through high-throughput screening of inhibitor compounds could lead to novel treatment strategies for H. pylori infections.
Comparative studies between YidC proteins from different Helicobacter species could also reveal species-specific adaptations that contribute to their distinct colonization patterns and disease associations. Such research would enhance our understanding of the evolutionary adaptations in membrane protein biogenesis systems across related bacterial pathogens.
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you require a specific format, please specify your preference during order placement. We will fulfill your request accordingly.
Note: All proteins are shipped with standard blue ice packs unless otherwise specified. If dry ice shipping is required, please contact us in advance. Additional fees may apply.
Generally, the shelf life for liquid form is 6 months at -20°C/-80°C. The shelf life for lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: heo:C694_07510
STRING: 85962.HP1450
Q&A
What is the genomic context of the yidC gene in Helicobacter pylori?
The yidC gene in H. pylori, like in other Gram-negative bacteria, is located within a highly conserved gene cluster. The specific gene order is rpmH, rnpA, yidD, yidC, and trmE . This conservation suggests functional importance of this arrangement. In E. coli, yidD overlaps with rnpA by 37 bp and is positioned only 2 bp upstream of yidC . The proximity and conservation of this gene organization indicates possible co-regulation or functional relationships between these genes. When designing gene knockout experiments, researchers should consider these overlaps to avoid polar effects on downstream gene expression.
How does YidC function in membrane protein insertion?
YidC operates through multiple pathways to facilitate membrane protein insertion:
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Sec-dependent pathway: YidC works in conjunction with the Sec translocon, assisting in the lateral movement and folding of transmembrane segments after they emerge from the SecYEG channel .
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YidC-only pathway: For certain substrates, YidC alone is sufficient for complete insertion and assembly into the inner membrane. These substrates include small coat proteins of the M13 and Pf3 phages, subunits a and c of the F₁F₀ ATPase, and subunit K of the NADH dehydrogenase complex .
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Hybrid pathway: Some proteins, like CyoA (subunit II of cytochrome bo₃ oxidase), require a mixed approach where YidC catalyzes insertion of the N-terminal domain while the Sec translocon is needed for translocation of the large C-terminal domain .
Methodologically, researchers should design experiments that can distinguish between these different pathways when studying novel YidC substrates in H. pylori.
What structural elements are critical for YidC function?
The hydrophobic slide consisting of transmembrane segments TM3 and TM5 forms a crucial structural feature of YidC that facilitates membrane protein insertion . Principal component analysis of YidC conformational dynamics reveals significant mobility in these regions during the insertion process . When designing mutations to study YidC function:
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Target residues within the hydrophobic slide to assess their role in substrate recognition
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Consider the conformational flexibility of YidC during insertion
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Examine the amphipathic helices that may facilitate membrane association
How do YidC conformational dynamics influence substrate insertion?
Principal component analysis (PCA) of YidC reveals significant conformational changes during membrane protein insertion. Analysis of different insertion poses (pose1 and pose2) shows that:
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PC1 and PC2 contributed 64.4% and 47.9% of the total variance, respectively
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The two poses demonstrate contradictory structural behaviors along PC1 and PC2
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YidC undergoes global and local structural rearrangements during the insertion process
| PC Component | Variance Contribution | Key Observation |
|---|---|---|
| PC1 | 64.4% | Clear differentiation between pose1 and pose2 |
| PC2 | 47.9% | Significant conformational differences |
Methodologically, researchers should employ molecular dynamics simulations with sufficient sampling to capture these conformational transitions when studying substrate-specific insertion mechanisms.
What is the role of YidD in YidC-mediated membrane protein insertion?
YidD is a small protein encoded by a gene that overlaps with rnpA and is positioned just 2 bp upstream of yidC . Though not essential for viability, experimental evidence indicates that YidD:
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Localizes to the inner membrane, likely through an amphipathic helix in its N-terminal region
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Affects the insertion and processing of YidC-dependent inner membrane proteins
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Can be cross-linked to nascent inner membrane proteins during their localization in the Sec-YidC translocon
For comprehensive studies of YidC function, researchers should consider the potential auxiliary role of YidD. Experimental approaches should include:
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Creating clean yidD knockout strains to assess its impact on YidC-mediated insertion
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Conducting in vitro reconstitution experiments with and without YidD
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Performing proximity labeling to identify interaction partners of YidD during membrane protein insertion
How does natural competence in H. pylori affect yidC evolution and function?
H. pylori is naturally competent for DNA uptake, which facilitates genome diversification through horizontal gene transfer . This characteristic may impact yidC evolution through:
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Homologous recombination: H. pylori harbors major recombination pathways that could facilitate allelic exchange of yidC variants during mixed infections
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Adaptive evolution: The high recombination rate might enable rapid adaptation of YidC to new substrates or environmental conditions
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Strain variation: Differences in YidC sequence or expression between H. pylori strains may correlate with pathogenicity or host adaptation
When studying YidC across H. pylori isolates, researchers should:
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Perform comparative genomic analyses to identify conserved and variable regions
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Correlate sequence variations with functional differences
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Consider the evolutionary pressure on YidC in the context of H. pylori's unique niche in the human stomach
What are the optimal expression systems for recombinant H. pylori YidC?
When expressing recombinant H. pylori YidC, consider these methodological approaches:
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Expression host selection:
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E. coli C41(DE3) or C43(DE3) strains are typically preferable for membrane protein expression
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Consider using H. pylori-optimized codons to improve expression
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For functional studies, complementation of E. coli YidC-depletion strains can verify activity
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Purification strategy:
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Include a detergent screening step (DDM, LMNG, or GDN often work well for membrane insertases)
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Implement a two-step purification protocol using affinity chromatography followed by size exclusion
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Verify protein folding using circular dichroism before functional assays
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Functional verification:
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In vitro translation/insertion assays with model substrates
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Complementation of yidC-depleted strains
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Substrate cross-linking studies to verify binding capabilities
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How can researchers assess YidC-substrate interactions in H. pylori?
Multiple complementary approaches should be employed to comprehensively characterize YidC-substrate interactions:
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In vivo cross-linking:
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Site-specific incorporation of photo-reactive amino acids at predicted interaction sites
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Use of chemical crosslinkers with membrane-permeable properties
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Analysis of crosslinked products using mass spectrometry to identify interaction sites
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In vitro reconstitution:
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Reconstitute purified YidC into liposomes or nanodiscs
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Add in vitro translated substrates to assess insertion efficiency
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Measure insertion using protease protection assays or fluorescence-based techniques
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Computational prediction:
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Molecular dynamics simulations to identify potential binding sites
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Sequence conservation analysis across H. pylori strains to identify functionally important residues
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Docking studies with known substrates to guide experimental design
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What techniques are effective for monitoring YidC-mediated membrane insertion in real-time?
Real-time monitoring of membrane insertion provides valuable kinetic insights:
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Fluorescence-based approaches:
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Position FRET pairs on YidC and substrate to monitor binding and insertion events
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Use environment-sensitive fluorophores that change signal upon membrane insertion
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Single-molecule FRET to capture insertion intermediates
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Electrical recording methods:
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Planar lipid bilayer recordings to capture conductance changes during insertion
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Solid-state nanopores modified with lipid bilayers for high-throughput measurements
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Patch-clamp of proteoliposomes containing reconstituted YidC
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Surface-sensitive techniques:
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Surface plasmon resonance to monitor binding kinetics
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Quartz crystal microbalance with dissipation to measure mass and conformational changes
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Atomic force microscopy to visualize topographical changes during insertion
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How does H. pylori YidC structural conformation compare to homologs in other bacteria?
H. pylori YidC shares the core structural elements with other bacterial homologs but exhibits specific adaptations:
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Conserved core structure:
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Species-specific adaptations:
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H. pylori YidC may have evolved specific features to function in the acidic environment of the stomach
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Differences in substrate specificity correlate with structural variations in the substrate-binding pocket
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Comparative analysis reveals conservation of functional residues despite sequence divergence
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Methodologically, researchers should:
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Perform homology modeling based on existing YidC structures
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Validate models using cross-linking and mutagenesis data
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Consider the impact of H. pylori's acidic environment on YidC structure and function
What approaches are most effective for resolving YidC-substrate complexes?
Resolving the structure of YidC-substrate complexes presents significant challenges that can be addressed through:
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Cryo-electron microscopy:
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Use of amphipols or nanodiscs to stabilize membrane protein complexes
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Application of focused refinement on the substrate-binding region
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Time-resolved studies to capture insertion intermediates
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X-ray crystallography:
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Co-crystallization with substrate fragments or designed antibodies to stabilize complexes
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Lipidic cubic phase crystallization to maintain native-like environment
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Surface engineering to promote crystal contacts while preserving functional interfaces
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Integrated structural biology:
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Combine low-resolution cryo-EM maps with molecular dynamics flexible fitting
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Validate models using crosslinking mass spectrometry data
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Use solid-state NMR to resolve specific interaction sites
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How does YidC contribute to H. pylori colonization and virulence?
YidC likely plays multiple roles in H. pylori pathogenesis through its essential function in membrane protein biogenesis:
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Virulence factor assembly:
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YidC may facilitate insertion of adhesins and other colonization factors
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Proper assembly of acid resistance proteins depends on YidC function
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Secretion systems required for host interaction may require YidC for component assembly
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Stress adaptation:
Experimental approaches to evaluate YidC's role in pathogenesis should include:
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Conditional yidC mutants to assess colonization efficiency in animal models
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Proteomic analysis of membrane protein composition under yidC depletion
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Identification of pathogenesis-specific YidC substrates
How do recombination and repair pathways in H. pylori impact YidC evolution?
H. pylori possesses unique DNA recombination and repair pathways that influence its genome plasticity :
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Natural competence system:
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Repetitive sequences:
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Strain diversity impact:
When studying YidC evolution in H. pylori, researchers should:
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Analyze synonymous vs. non-synonymous substitution rates to identify selection pressures
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Examine population structure of yidC alleles across clinical isolates
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Correlate YidC sequence variants with functional differences and pathogenicity
