Recombinant Enterobacteria phage If1 Head virion protein G6P (VI)
Description
Introduction to Enterobacteria Phage If1
Enterobacteria phage If1 is a filamentous bacteriophage that targets bacteria with I pili, particularly Escherichia coli. Like other filamentous phages, If1 contains several structural proteins that play crucial roles in its infection cycle and viral architecture . The phage demonstrates structural and functional similarities to other well-characterized filamentous phages, though with distinct targeting mechanisms.
General Structure and Composition
Filamentous bacteriophages like If1 typically possess a protein coat surrounding their single-stranded DNA genome. These viruses have evolved specialized infection mechanisms that begin with the recognition of specific receptors on the bacterial cell surface. In the case of phage If1, the virus specifically targets I pili on the bacterial surface, which contrasts with related phages like fd that target F pili .
Key Proteins in Enterobacteria Phage If1
Current research indicates that Enterobacteria phage If1 contains several important structural and functional proteins. Among the characterized proteins are Gene 1 protein (G1P) and Gene 3 protein (G3P), both of which have been studied in recombinant form . The Gene 3 protein (G3P) plays a particularly crucial role in host recognition and attachment .
While the available data focuses primarily on G1P and G3P proteins, the virion structure likely includes additional proteins that contribute to phage structure and function, potentially including head virion proteins similar to the G6P found in related phages.
Function and Importance of Head Virion Proteins
Head virion proteins in bacteriophages generally serve critical structural and functional roles. These proteins typically form part of the capsid or head structure that encapsulates the viral genetic material. Additionally, they may participate in host recognition, attachment, and infection processes essential for viral replication.
Comparative Analysis with Related Phages
While specific information about the G6P (VI) protein in phage If1 is limited in the provided sources, related filamentous phages such as M13 are known to contain head virion protein G6P (VI) . In phage M13, this protein contributes to the virion structure and participates in the assembly and stability of the viral particle.
The presence of similar structural components across related phages suggests that phage If1 likely contains analogous proteins that perform comparable functions. Based on evolutionary conservation patterns observed in filamentous phages, it is reasonable to infer that If1 G6P would share structural and functional characteristics with its counterparts in other phages.
Protein Structure and Domains
The detailed molecular structure of Enterobacteria phage If1 proteins provides insights into their function. For example, the G3P protein of phage If1 contains N1 and N2 domains that function independently, with N2 mediating binding to the I pilus and N1 targeting the bacterial TolA protein . This domain organization differs from that observed in related phages like fd, where the domains interact closely with each other.
Expression Systems and Methodologies
Recombinant phage proteins are typically produced using bacterial expression systems, with E. coli being the most common host . These systems allow for the addition of affinity tags, such as the His-tag observed in recombinant G1P, which facilitate purification through affinity chromatography.
Purification and Characterization Methods
The purification of recombinant phage proteins generally follows established protocols for protein isolation and characterization. For example, recombinant G1P from phage If1 is produced as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE . Standard storage conditions include -20°C/-80°C storage with recommendations to avoid repeated freeze-thaw cycles.
Phage If1 Infection Process
Research on phage If1 has revealed a distinct infection mechanism compared to related phages. While phage fd uses a two-step process involving domain unfolding and prolyl isomerization for infection, phage If1 employs a different approach. In If1, the domains of G3P function independently, with the TolA binding site on N1 remaining permanently accessible without requiring activation through unfolding or isomerization .
Domain Organization and Functional Implications
The interaction between phage If1 G3P and its bacterial receptor demonstrates evolutionary adaptation. Unlike phage fd, where the N1 and N2 domains are tightly associated and require structural changes for activation, If1 G3P has independently folding units. This structural difference is compensated by increased stability of the individual domains, indicating alternative evolutionary strategies to balance robustness with infectivity .
Biotechnological Applications
Recombinant bacteriophage proteins have diverse applications in biotechnology, including as research tools, diagnostic reagents, and potential therapeutic agents. The availability of recombinant versions of phage If1 proteins facilitates these applications by providing purified material for experimental use.
Phage Display and Protein Engineering
Understanding the structure and function of phage proteins enables their use in techniques such as phage display, which has applications in antibody discovery, protein engineering, and targeted drug delivery. The distinct characteristics of phage If1 proteins could offer advantages for specific applications in these fields.
Comparative Viral Studies
The structural and functional differences between related phages like If1 and fd provide valuable insights into viral evolution and adaptation strategies. These comparisons enhance our understanding of how viruses optimize their infection mechanisms while maintaining structural integrity.
Current Limitations in Knowledge
While significant progress has been made in characterizing certain proteins from phage If1, such as G1P and G3P, detailed information about other components, including the head virion protein G6P (VI), remains limited. This knowledge gap presents opportunities for further research to fully characterize the complete protein complement of phage If1.
Recommended Research Approaches
Future studies should focus on:
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Complete structural characterization of all phage If1 proteins
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Functional analysis of head virion proteins, including G6P
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Comparative studies with related phages to identify conserved and divergent features
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Investigation of potential applications in biotechnology and medicine
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them when placing your order. We will accommodate your request whenever possible.
Note: All of our proteins are shipped standard with blue ice packs. If you require dry ice shipment, please communicate this to us in advance. Additional fees will apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C. Lyophilized forms typically have a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: vg:1261856
Q&A
What is the molecular identity of Enterobacteria phage If1 Head virion protein G6P(VI)?
Enterobacteria phage If1 Head virion protein G6P(VI) is a structural protein from Bacteriophage If1 with UniProt identifier O80298 . The full-length protein consists of 113 amino acids with the sequence: MPVFLGLPVLARFIGWLAGALIAYVAKFFTLGIARIALAISLFLGLIIGLNGLLVSYLSDLTSVLPPEIASAVSYVVPANAAPCLYAIFSLKAAVFIFDVKDRIIGYLDWNKS . This protein functions as a component of the phage head structure and is essential for proper virion assembly.
What role does G6P(VI) play in bacteriophage structure and assembly?
The G6P(VI) protein serves as a structural component in the bacteriophage head assembly. In general, phage head assembly involves three essential components: capsid proteins, scaffolding proteins, and portal proteins . While the search results don't specify the exact function of G6P(VI), head virion proteins typically form part of the capsid structure that protects the viral genetic material. The assembly process likely involves copolymerization of scaffolding and capsid proteins initiated from the portal vertex . As a structural protein, G6P(VI) contributes to the formation of the stable, mature phage head structure necessary for successful phage replication and infection.
How is recombinant G6P(VI) typically stored and handled in laboratory settings?
Recombinant G6P(VI) is optimally stored in a Tris-based buffer containing 50% glycerol at -20°C for regular storage, or at -80°C for extended storage periods . Working aliquots can be maintained at 4°C for up to one week . Repeated freezing and thawing cycles should be avoided to maintain protein integrity and activity . The specific buffer composition has been optimized for this particular protein to ensure stability and functionality during storage and experimental use.
What experimental approaches are most effective for studying G6P(VI) structure-function relationships?
For studying structure-function relationships of G6P(VI), researchers should consider employing a multi-faceted approach combining:
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X-ray crystallography: This technique has been successfully applied to determine the structure of portal proteins in phages like Sf61989, suggesting its applicability for G6P(VI) .
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Cryo-electron microscopy (cryo-EM): Cryo-EM has been effectively used to visualize phage head structures and the arrangement of dsDNA within the capsid . This technique would allow visualization of G6P(VI) in its native context within the assembled phage head.
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Single-molecule studies: Similar to those performed for the phi29 packaging motor, these can provide insights into dynamic conformational changes during assembly .
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Proteomic mass spectrometry: This approach has been successfully used to confirm the production of hypothetical proteins in Vi01-like phages, revealing unexpected functions and associations .
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Mutational analysis: Specific mutations can be introduced to assess the impact on head assembly and stability, similar to studies conducted with other phages like T4, P22, and SPP1 .
How can machine learning approaches be applied to study G6P(VI) and related virion proteins?
Machine learning approaches offer powerful tools for studying phage virion proteins like G6P(VI). The RF_phage virion method represents an effective classification approach for virion proteins using four protein sequence coding methods as features :
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Amino Acid Composition (AAC): Analyzes the frequency of each amino acid in the protein sequence.
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CSKAAP: Captures composition, transition, and distribution of amino acid properties.
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Dipeptide Composition (DPC): Examines the frequency of dipeptides in the sequence.
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Dipeptide Deviation from Expected mean (DDE): Measures the deviation of observed dipeptide frequencies from expected values .
These features can be combined with random forest algorithms to create highly accurate classification models. For G6P(VI) specifically, these approaches could help predict functional domains, interaction sites, or evolutionary relationships with other phage proteins . The performance metrics of the combined feature approach have demonstrated superior classification accuracy compared to individual feature methods .
What is known about the conformational dynamics of head virion proteins during phage maturation?
Phage head maturation involves substantial conformational changes that transform the prohead into a mature, infectious virion. During or before DNA packaging, scaffolding proteins either exit from the capsid (as in phages P22 and phi29) or are proteolytically cleaved by phage-encoded proteases (as in HK97 and T4) . This process initiates maturation, involving a large structural transition of the prohead and resulting in a larger, more angular, and more stable head with a thinner shell .
Head expansion is likely initiated at one end by the portal protein and then propagates through the prohead, resulting in approximately a 50% increase in head volume . This expansion leads to changes in the appearance of hexameric capsomers, which in most phages have two-fold rather than six-fold symmetry in the prohead . In phage P22, skewed capsomers have central holes through which scaffolding proteins could exit .
For G6P(VI) specifically, understanding its role in this maturation process would require targeted studies examining its conformational changes during the assembly and maturation process.
What are the optimal protocols for expression and purification of recombinant G6P(VI)?
While the search results don't provide specific protocols for G6P(VI) expression and purification, general approaches for recombinant phage proteins can be adapted:
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Expression system selection: Based on the amino acid sequence of G6P(VI), which contains both hydrophobic and hydrophilic regions , a bacterial expression system like E. coli with appropriate fusion tags would likely be suitable.
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Purification strategy: A multi-step purification approach typically involving:
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Initial affinity chromatography using an appropriate tag
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Ion exchange chromatography for further purification
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Size exclusion chromatography as a final polishing step
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Buffer optimization: The final storage buffer for G6P(VI) (Tris-based with 50% glycerol) suggests that the protein may have stability issues that are addressed by the high glycerol content. During purification, buffers should be optimized to maintain protein solubility and prevent aggregation.
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Quality control: Final purified protein should be assessed using SDS-PAGE, Western blotting, and mass spectrometry to confirm identity and purity.
How can researchers investigate G6P(VI) interactions with other phage components?
To investigate G6P(VI) interactions with other phage components, researchers should consider:
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Co-immunoprecipitation assays: These can identify protein-protein interactions between G6P(VI) and other phage proteins.
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Pull-down assays: Using tagged versions of G6P(VI) to identify binding partners.
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Surface plasmon resonance (SPR): For quantitative measurement of binding affinities between G6P(VI) and other phage components.
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Cross-linking mass spectrometry: This approach can identify specific interaction sites between G6P(VI) and other proteins in the phage assembly.
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Cryo-EM of assembled structures: To visualize G6P(VI) in the context of the complete virion structure, similar to approaches used for other phage head proteins .
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DNA-protein interaction assays: If G6P(VI) potentially interacts with DNA, techniques like electrophoretic mobility shift assay (EMSA) could be employed to characterize these interactions, similar to studies done with other phage head proteins and DNA .
How does G6P(VI) compare structurally and functionally to similar proteins in other phage families?
Head virion proteins across different phage families share similar functional roles but can vary significantly in sequence and structure. The table below compares key features of G6P(VI) with similar proteins from other phage families:
This comparative analysis highlights the diversity of head assembly mechanisms across different phage families. While some general principles are conserved (such as the role of scaffolding proteins and maturation processes), the specific molecular details vary considerably.
What bioinformatic approaches are most effective for analyzing G6P(VI) sequence and predicting its function?
For effective bioinformatic analysis of G6P(VI), researchers should employ:
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Sequence homology searches: Using tools like BLAST to identify related proteins across phage families.
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Structural prediction: Employing tools like AlphaFold or RoseTTAFold to predict the three-dimensional structure of G6P(VI).
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Domain identification: Using databases like Pfam and PROSITE to identify functional domains within the protein.
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Machine learning classification: The RF_phage virion approach using multiple feature extraction methods (AAC, CSKAAP, DPC, and DDE) has demonstrated high accuracy in classifying phage virion proteins . This approach could help predict functional aspects of G6P(VI).
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Evolutionary analysis: Phylogenetic comparisons with similar proteins from other phages to understand evolutionary relationships and functional conservation.
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Protein-protein interaction prediction: Using computational methods to predict potential interaction partners within the phage proteome.
The combination of these approaches would provide a comprehensive understanding of G6P(VI)'s potential functions and interactions within the phage assembly.
How can G6P(VI) be utilized in phage biology and biotechnology research?
G6P(VI) has several potential applications in phage biology and biotechnology research:
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Structural biology studies: As a component of the phage head, G6P(VI) can serve as a model for understanding protein-protein interactions in viral assembly.
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Phage engineering: Knowledge of G6P(VI) structure and function could enable engineering of phages with modified capsid properties for applications in phage therapy or phage display.
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Bionanotechnology: Phage head proteins like G6P(VI) can potentially be used to create self-assembling nanostructures for various applications.
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Host-range studies: Understanding the role of virion proteins in host specificity could help develop phages with tailored host ranges, similar to studies done with Vi01-like phages .
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Diagnostic applications: Proteins like G6P(VI) could be developed into tools for detecting and characterizing bacterial pathogens targeted by the phage.
What role might G6P(VI) play in understanding broader mechanisms of viral assembly?
G6P(VI) research could contribute significantly to understanding broader mechanisms of viral assembly in several ways:
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Assembly initiation: Studies of phage head proteins have revealed that assembly is often initiated from the portal vertex by copolymerization of scaffolding and capsid proteins . G6P(VI) research could provide insights into this process.
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Maturation processes: Understanding how G6P(VI) participates in the transition from prohead to mature head could illuminate viral maturation mechanisms more broadly.
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Protein-protein interactions: Characterizing G6P(VI) interactions with other phage components could reveal conserved principles of viral assembly.
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Conformational changes: Studying conformational changes in G6P(VI) during assembly could provide insights into how structural proteins contribute to the dynamic process of viral assembly.
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DNA packaging mechanisms: If G6P(VI) interacts with DNA, it could provide insights into how viruses package their genetic material, a process that has been studied extensively in phages like T4, P22, and SPP1 .
