Recombinant Pseudomonas phage Pf3 Head virion protein G6P (VI)

How does recombinant G6P differ from native phage-derived G6P in terms of structure and function?

Recombinant G6P protein produced in heterologous expression systems may exhibit subtle differences from native phage-derived G6P. These differences typically arise from:

• Post-translational modifications: Bacterial expression systems may lack the machinery for specific modifications present in the native host

• Folding variations: Environmental conditions during recombinant expression might affect protein folding

• Functional effects: Recombinant proteins may show altered activity or stability profiles

Methodologically, researchers can compare these differences using structural techniques such as circular dichroism, limited proteolysis, and thermal shift assays. Functional analysis often involves assembly assays to test the ability of recombinant G6P to form virus-like particles or complement phage assembly mutants. Structural integrity can be verified using approaches similar to those used for studying other phage proteins, including cryo-EM techniques at high resolution (3-4 Å) that have been successfully applied to phages like E217 .

What are the key structural features of Pseudomonas phage head proteins identified through cryo-EM and other structural techniques?

Structural analysis of Pseudomonas phage head proteins has revealed several conserved features, though specific details for Pf3 G6P are not extensively documented in the current literature. Based on studies of related phage systems:

• Many phage head proteins adopt HK97-like folds with characteristic β-sheet domains

• These proteins often assemble into capsomers that form pentamers at the vertices and hexamers across the faces of the icosahedral capsid

• Surface decorations or protrusions may be present, like those formed by the trimeric gp24 protein observed in phage E217

High-resolution cryo-EM has become the method of choice for structural determination of phage components, allowing visualization at near-atomic resolution. For instance, the E217 phage head was reconstructed using localized reconstruction with fivefold symmetry imposed (C5), yielding 2.8 Å resolution . This approach combined with local symmetry averaging can reveal intricate structural details of capsid proteins and their interactions.

What are the optimal expression systems for producing high yields of functional recombinant Pseudomonas phage Pf3 G6P protein?

The optimal expression systems for recombinant Pseudomonas phage proteins typically include:

For optimal expression, researchers should consider using specialized vectors containing appropriate promoters (T7, tac) and fusion tags (His6, MBP, GST) to facilitate purification. Expression conditions should be optimized through systematic testing of induction parameters (temperature: 16-30°C; IPTG concentration: 0.1-1.0 mM; induction time: 4-24 hours). This methodological approach parallels successful strategies used for other recombinant phage proteins and enzymes, such as those described for the fused TvG6PD::6PGL protein .

What purification challenges are commonly encountered with recombinant Pseudomonas phage head proteins, and how can they be addressed?

Common purification challenges for recombinant phage head proteins include:

• Solubility issues: Phage structural proteins often form inclusion bodies in heterologous expression systems

• Aggregation: Head proteins may aggregate during concentration steps

• Co-purification of host contaminants: Bacterial proteins may bind non-specifically to purification resins

Methodological approaches to address these challenges include:

• For solubility: Optimize expression temperature (often lowering to 16-18°C), co-express with chaperones, or use solubility-enhancing fusion partners like MBP

• For aggregation: Include stabilizing agents (5-10% glycerol, 100-250 mM NaCl) in all buffers, avoid freeze-thaw cycles

• For purification: Implement multi-step chromatography protocols combining affinity chromatography (Ni-NTA for His-tagged proteins) followed by ion exchange and size exclusion chromatography

For instance, successful purification strategies for other recombinant proteins often employ affinity chromatography as demonstrated with the TvG6PD::6PGL protein, which was efficiently purified using a similar approach .

How can researchers assess the proper folding and stability of recombinant G6P protein after purification?

Assessment of proper folding and stability of recombinant G6P protein can be accomplished through a multi-technique approach:

• Thermal stability analysis:

  • Differential scanning fluorimetry (DSF/Thermofluor) to determine melting temperature (Tm)

  • Circular dichroism (CD) thermal denaturation to monitor secondary structure changes

• Structural integrity evaluation:

  • Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to assess oligomeric state

  • Limited proteolysis to verify compact folding (properly folded proteins typically show resistance to proteolytic digestion)

• Functional assays:

  • Assembly competence tests to verify ability to form higher-order structures

  • Binding assays with known interaction partners (e.g., other capsid proteins)

Similar approaches have been successfully applied to other recombinant proteins, such as the assessment of TvG6PD::6PGL, where researchers evaluated temperature effects, susceptibility to trypsin digestion, and stability in the presence of guanidine hydrochloride with and without substrates/cofactors . For G6P specifically, researchers should consider evaluating stability under conditions that mimic the phage assembly environment.

What experimental approaches can determine the role of G6P in phage assembly and infection processes?

Experimental approaches to determine G6P's role in phage assembly and infection include:

• Genetic manipulation studies:

  • Deletion or mutation of the G6P gene to assess effects on phage viability

  • Complementation assays to verify functional restoration

• In vitro assembly systems:

  • Reconstitution of capsid assembly using purified components

  • Electron microscopy to visualize assembly intermediates and final structures

• Interaction mapping:

  • Pull-down assays to identify binding partners within the phage proteome

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

• Host interaction studies:

  • Phage adsorption assays with wild-type versus G6P-mutant phages

  • Time-course infection experiments monitored by electron microscopy

How do post-translational modifications affect the function of recombinant Pseudomonas phage head proteins?

Post-translational modifications (PTMs) can significantly impact phage head protein function in various ways:

To systematically study the impact of PTMs:

• Compare native and recombinant proteins using mass spectrometry to identify differences in modification patterns

• Generate site-directed mutants at modification sites to assess functional consequences

• Perform in vitro modification assays to determine if artificially introducing specific PTMs restores function

For phage structural proteins like G6P, the most relevant PTMs likely involve proteolytic processing and disulfide bond formation, which can be critical for proper folding and assembly. Researchers should consider expression systems that support these modifications when producing recombinant G6P for functional studies.

What biophysical techniques are most informative for studying G6P interactions with other phage structural proteins?

Several biophysical techniques provide valuable insights into G6P interactions with other phage structural proteins:

• Surface Plasmon Resonance (SPR) and Bio-Layer Interferometry (BLI):

  • Determine binding kinetics and affinity constants (Ka, Kd)

  • Identify binding dependencies on buffer conditions, pH, and ionic strength

• Isothermal Titration Calorimetry (ITC):

  • Measure thermodynamic parameters (ΔH, ΔS, ΔG)

  • Determine stoichiometry of interactions

• Microscale Thermophoresis (MST):

  • Analyze interactions in solution with minimal protein consumption

  • Detect subtle conformational changes upon binding

• Structural methods:

  • Cryo-EM of assembled complexes at high resolution (3-4 Å)

  • Cross-linking mass spectrometry (XL-MS) to identify interaction interfaces

These techniques should be applied sequentially, starting with screening methods (like MST) followed by more detailed analysis (SPR/BLI, ITC) and structural confirmation. The high-resolution structural approach used for E217 phage (3.1-4.5 Å resolution cryo-EM) represents an excellent methodological template for studying G6P in the context of the Pf3 phage head.

How can computational modeling be used to predict functional sites and structural dynamics of Pseudomonas phage Pf3 G6P?

Computational modeling offers powerful approaches for predicting functional sites and structural dynamics of phage proteins like G6P:

• Homology modeling and threading:

  • Generate 3D structural models based on related phage proteins with known structures

  • Validate models using quality assessment tools (QMEAN, ProSA)

• Molecular dynamics (MD) simulations:

  • Explore conformational dynamics on nanosecond to microsecond timescales

  • Identify flexible regions and stable structural cores

• Functional site prediction:

  • Identify conserved residues through multiple sequence alignment of related phage proteins

  • Use tools like ConSurf, COACH, and FTSite to predict functional interfaces

• Protein-protein docking:

  • Model interactions with other capsid components

  • Perform ensemble docking to account for conformational flexibility

Similar computational approaches have been successfully applied to other proteins, as demonstrated in the study of TvG6PD::6PGL where researchers generated a 3D model to understand its structural features despite limited sequence homology with human proteins . For G6P, researchers could employ similar methods, starting with template identification, model generation, and refinement, followed by validation and functional annotation.

What are the current hypotheses regarding the evolutionary relationships between Pseudomonas phage head proteins and similar proteins in other phage families?

Current evolutionary hypotheses regarding phage head proteins focus on several key aspects:

• Structural conservation despite sequence divergence:

  • Many phage head proteins share the HK97-like fold despite minimal sequence identity

  • This suggests strong structural constraints during evolution

• Modular evolution:

  • Head proteins often evolve as functional modules that can be exchanged between phages

  • Sequence analysis reveals evidence of horizontal gene transfer between phage lineages

• Host-adaptation signatures:

  • Head proteins show adaptation to specific bacterial hosts, particularly in surface-exposed regions

  • Codon usage patterns often reflect adaptation to host translation machinery

To investigate these hypotheses for G6P specifically, researchers should:

• Conduct comprehensive phylogenetic analysis comparing G6P with head proteins from diverse phage families

• Perform structural superposition of G6P models with experimentally determined structures like those from E217

• Analyze patterns of sequence conservation in the context of predicted structural features

These approaches can reveal whether G6P represents a conserved structural component or has unique features specific to Pseudomonas phages.

How might structural insights from high-resolution cryo-EM studies of G6P inform the development of phage-based antimicrobials against Pseudomonas infections?

High-resolution structural studies of G6P could significantly inform antimicrobial development through several research directions:

• Identification of critical assembly interfaces:

  • Mapping residues essential for capsid formation

  • Developing inhibitors that disrupt phage assembly as tools for studying phage biology

• Host-recognition mechanisms:

  • Understanding structural features involved in host specificity

  • Engineering modified phages with expanded or altered host range

• Stability determinants:

  • Identifying structural elements contributing to environmental stability

  • Enhancing phage stability for therapeutic applications

• Rational phage engineering:

  • Designing chimeric phages with optimized properties

  • Creating platform technologies for delivering antimicrobial payloads

The methodological approach would parallel high-resolution cryo-EM studies of phages like E217, which revealed critical structural information about capsid assembly and host interaction mechanisms . For G6P specifically, researchers could apply similar techniques to determine its structure at comparable resolution (3-4 Å) and integrate this information into a comprehensive structural model of the Pf3 phage capsid.

What technical challenges exist in applying advanced structural methods like cryo-EM and X-ray crystallography to Pseudomonas phage head proteins, and how can they be overcome?

Several technical challenges exist in structural studies of phage head proteins, with corresponding methodological solutions:

For cryo-EM specifically, researchers studying G6P should consider:

• Optimizing sample preparation with appropriate detergents or nanodiscs if membrane interactions are present

• Applying multiple symmetry assumptions during processing to identify true symmetry

• Using advanced particle classification to separate different conformational states

• Implementing focused refinement strategies as demonstrated in the E217 study

These approaches can help overcome the inherent challenges of working with complex phage structural proteins and yield high-resolution structural information.

How might synthetic biology approaches be used to engineer novel functions into Pseudomonas phage Pf3 head proteins?

Synthetic biology offers several promising approaches for engineering novel functions into phage head proteins like G6P:

• Modular design strategies:

  • Identify functionally independent domains through structural analysis

  • Create fusion proteins incorporating domains with novel functions

• Display technology:

  • Engineer G6P to display peptides or proteins on the phage surface

  • Develop phage display libraries using G6P as a scaffold

• Capsid modification for cargo delivery:

  • Introduce interior modifications to allow encapsulation of non-native cargo

  • Engineer controlled release mechanisms triggered by specific stimuli

• Structural stabilization:

  • Introduce disulfide bonds or salt bridges to enhance stability

  • Apply computational design to optimize interfaces between capsid subunits

The methodological approach would include:

• Structure-guided design based on high-resolution models

• Iterative testing using recombinant expression and functional assays

• In vitro assembly tests to verify capsid formation

• Verification of novel functions using appropriate bioassays

These approaches would build upon structural insights from studies like those conducted on E217 phage , applying similar principles to engineer new functions into the G6P protein of Pf3 phage.

What is the current understanding of the atomic-level mechanisms of Pseudomonas phage head assembly, and what critical questions remain unanswered?

Current understanding of Pseudomonas phage head assembly at the atomic level reveals several key mechanisms, though many questions remain:

Current knowledge:

• Many phage capsids assemble through a procapsid intermediate that undergoes maturation

• Portal complexes often serve as nucleation points for assembly, creating asymmetry at one vertex

• Chaperones and scaffolding proteins frequently guide the assembly process

• Head completion proteins seal the capsid after DNA packaging

Critical unanswered questions for G6P and related proteins:

• What triggers the transition from the procapsid to mature capsid state?

• How is assembly fidelity maintained to prevent aberrant structures?

• What is the precise temporal sequence of protein incorporation during assembly?

• How do head proteins recognize the correct vertex for tail attachment?

To address these questions, researchers should apply a combination of:

• Time-resolved cryo-EM to capture assembly intermediates

• Single-molecule techniques to track assembly kinetics

• Mass photometry to measure assembly stoichiometry

• In vitro reconstitution systems with purified components

The high-resolution structural approaches used for E217 phage provide an excellent methodological template for similar studies on the Pf3 phage head assembly process.

How do variations in G6P structure across different Pseudomonas phage strains correlate with host range and infectivity profiles?

The relationship between G6P structural variations and phage host range/infectivity represents an important area of investigation:

• Structural determinants of host specificity:

  • Surface-exposed regions of head proteins may interact with host factors

  • Variations in these regions could influence host recognition or evasion of host defenses

• Comparative analysis approach:

  • Sequence and structural comparison of G6P across Pseudomonas phages with different host ranges

  • Identification of variable regions that correlate with host specificity

• Experimental validation:

  • Generation of chimeric phages with G6P regions swapped between strains

  • Host range testing of engineered phages to confirm functional correlations

  • Binding assays with host components to identify interaction partners

• Structural biology integration:

  • High-resolution structural studies of G6P variants using cryo-EM

  • Mapping of variable regions onto structural models to identify surface-exposed differences

This research direction would benefit from applying similar methodological approaches to those used in studying the E217 phage, where high-resolution structural data revealed important host-recognition mechanisms . For G6P specifically, researchers should focus on identifying regions with high sequence variability and determining their structural context.