Recombinant Enterobacteria phage PRD1 Protein P18 (XVIII)

What is the structural composition of recombinant Enterobacteria phage PRD1 Protein P18 (XVIII)?

Recombinant P18 (XVIII) protein is a full-length (1-90 amino acids) integral membrane protein from Enterobacteria phage PRD1. The protein has a complete amino acid sequence of MPFGLIVIGIILAIAAYRDTLGELFSIIKDVSKDAKGFGYWVLAAVILGFAASIKPIKEPVNAFMILLMIVLLIRKRGAIDQISNQLRGS. When produced recombinantly, it is typically fused to an N-terminal His-tag and expressed in E. coli expression systems, resulting in a protein with greater than 90% purity as determined by SDS-PAGE . The protein is hydrophobic in nature, containing multiple membrane-spanning regions that integrate into the PRD1 viral membrane during natural assembly processes. Understanding this structural composition is essential for designing experiments involving membrane protein reconstitution and functional studies.

How should recombinant P18 (XVIII) protein be stored and handled in laboratory settings?

For optimal stability and activity, recombinant P18 (XVIII) protein should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles . The lyophilized protein powder is typically supplied in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 . For reconstitution, researchers should:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to 5-50% final concentration (50% is recommended) for long-term storage

• Store working aliquots at 4°C for up to one week only

It's important to note that repeated freezing and thawing is not recommended as it can compromise protein integrity and functional activity.

What expression systems are used for producing recombinant P18 protein, and what are their comparative advantages?

The primary expression system documented for producing recombinant P18 protein is E. coli . This prokaryotic system offers several advantages for membrane protein expression:

For functional studies of P18, E. coli expression is typically sufficient as the protein does not require complex post-translational modifications and comes from a bacterial phage environment .

What analytical methods are most effective for confirming the purity and integrity of recombinant P18 protein?

Several analytical methods can be employed to verify the purity and integrity of recombinant P18 protein:

• SDS-PAGE Analysis: The standard method for determining protein purity (>90% purity is typically achieved) . For membrane proteins like P18, specialized detergent-containing loading buffers may be necessary.

• Western Blotting: Using either anti-His antibodies or specific antibodies against P18, similar to methods described for other PRD1 proteins such as P16 . This method not only confirms identity but can also detect degradation products.

• Mass Spectrometry: Provides accurate molecular weight determination and can confirm the amino acid sequence.

• Circular Dichroism (CD) Spectroscopy: Useful for assessing secondary structure integrity, particularly important for membrane proteins.

• Size Exclusion Chromatography: Can determine if the protein exists as monomers or forms higher-order complexes.

For membrane proteins like P18, it's also important to assess proper folding in membrane-mimetic environments using techniques such as proteoliposome reconstitution followed by functional assays.

How does P18 (XVIII) integrate with other PRD1 structural proteins, and what methods can be used to study these interactions?

P18 (XVIII) is part of the complex machinery of PRD1's membrane structure. While the search results don't specifically detail P18's interactions, we can infer potential research approaches based on studies of related PRD1 membrane proteins:

• Co-immunoprecipitation (Co-IP): Using antibodies against P18 to pull down interacting partners, similar to techniques used to study other PRD1 vertex proteins .

• Yeast Two-Hybrid (Y2H) or Bacterial Two-Hybrid (B2H) Assays: May be used to screen for potential protein-protein interactions, though these can be challenging with membrane proteins.

• Proximity-Based Labeling: Techniques such as BioID or APEX2 fusion proteins can identify nearby proteins in the native viral environment.

• Cross-linking Mass Spectrometry (XL-MS): Can capture transient or stable interactions between P18 and other viral components.

• Cryo-Electron Microscopy: For structural determination of P18 within the context of the viral particle.

Drawing parallels with other PRD1 membrane proteins, P18 might interact with vertex components similar to how P16 links the spike complex to the viral membrane , or like P20 and P22 which connect the packaging machinery to the viral membrane . Experimental designs should consider the integral membrane nature of P18 and the need for appropriate detergents or lipid environments.

What role does P18 (XVIII) play in PRD1 viral assembly and infection compared to other membrane proteins such as P16?

The specific role of P18 (XVIII) is not explicitly detailed in the search results, but comparative analysis with other PRD1 membrane proteins allows for informed hypotheses:

To investigate P18's specific role, researchers could:

• Generate P18-deficient mutants through amber mutations or CRISPR-based approaches in bacterial hosts carrying PRD1

• Analyze structural integrity of resulting viral particles

• Conduct complementation studies with wild-type P18

• Perform comparative infection assays and structural analyses

These approaches would help distinguish P18's role from that of other membrane proteins like P16, which has been shown to stabilize the spike complex at vertices .

What reconstitution methods are most effective for studying recombinant P18 protein in membrane-mimetic systems?

For functional and structural studies of membrane proteins like P18, several reconstitution methods can be employed:

• Detergent Micelles:

  • Use mild detergents (DDM, LDAO, OG)

  • Advantages: Simple preparation, good for initial solubilization

  • Limitations: May not fully recapitulate native membrane environment

• Liposome Reconstitution:

  • Preparation of proteoliposomes with defined lipid composition

  • Methods: Detergent dialysis, rapid dilution, or direct incorporation

  • Advantages: More native-like environment

  • Compatible with functional assays such as permeability studies

• Nanodiscs:

  • Using membrane scaffold proteins (MSPs) to create disc-like bilayers

  • Advantages: Defined size, access to both sides of membrane, compatibility with various biophysical techniques

  • Particularly useful for cryo-EM studies

• Bicelles:

  • Mixtures of long-chain and short-chain phospholipids

  • Advantages: Compatible with NMR studies

For P18 specifically, reconstitution should consider the lipid composition of the PRD1 membrane, which differs from host bacterial membranes. A systematic approach would involve:

• Initial solubilization in mild detergents

• Testing different lipid compositions for reconstitution

• Functional verification through assays relevant to hypothesized P18 functions

• Structural characterization in the reconstituted system

How can researchers differentiate between functional and structural roles of P18 in PRD1 viral membrane organization?

Distinguishing between functional and structural roles requires multi-faceted experimental approaches:

By correlating structural features with functional outcomes across these experimental approaches, researchers can delineate whether P18 serves primarily as a structural scaffolding protein or has specific functional roles in processes such as membrane reorganization during infection.

What biophysical techniques are most informative for characterizing the membrane topology and dynamics of P18 protein?

Several biophysical techniques can provide valuable insights into P18's membrane topology and dynamics:

A comprehensive characterization would combine multiple techniques, starting with computational predictions of membrane topology based on the known amino acid sequence (MPFGLIVIGIILAIAAYRDTLGELFSIIKDVSKDAKGFGYWVLAAVILGFAASIKPIKEPVNAFMILLMIVLLIRKRGAIDQISNQLRGS) , followed by experimental validation using the techniques above.

What are the critical control experiments needed when working with recombinant P18 protein in functional assays?

When designing experiments with recombinant P18 protein, several critical controls should be implemented:

• Protein Quality Controls:

  • Negative control: Empty vector-expressed preparations

  • Positive control: Known functional membrane protein expressed under identical conditions

  • Thermo-stability assessment: Pre-incubation at various temperatures to establish functional stability range

• Expression System Controls:

  • Comparison of P18 expressed in different systems (if available)

  • Assessment of post-expression modifications

• Reconstitution Controls:

  • Liposomes/nanodiscs without P18

  • Reconstitution with denatured P18

  • Reconstitution with varying P18:lipid ratios

• Functional Assay Specificity Controls:

  • Competitive inhibition with antibodies against P18

  • Comparison with known PRD1 membrane proteins (P16, P20, P22)

  • Site-directed mutants with altered predicted functional regions

• Biological Relevance Controls:

  • Complementation assays with P18-deficient PRD1 phage

  • Comparison of in vitro findings with in vivo phenotypes

These controls help distinguish specific P18 effects from artifacts and provide context for interpreting experimental results, especially important given the limited direct information available about P18's specific functions.

How can researchers effectively use recombinant P18 protein to study PRD1 membrane dynamics during viral infection?

Studying membrane dynamics during viral infection using recombinant P18 requires sophisticated experimental designs:

• Fluorescently Labeled P18:

  • Site-specific labeling at non-critical residues

  • Integration into artificial membrane systems

  • Real-time tracking during membrane transformation events

• Reconstituted Systems for Studying Membrane Transformations:

  • Creation of PRD1-like vesicles with defined protein composition

  • Monitoring membrane curvature changes under varying conditions

  • Assessing P18 redistribution during membrane reorganization

• High-Resolution Microscopy Approaches:

  • Single-molecule tracking of labeled P18

  • Super-resolution microscopy to visualize P18 clustering

  • Correlative light and electron microscopy to connect dynamics with ultrastructure

• Biomimetic Platforms:

  • Supported lipid bilayers with incorporated P18

  • Microfluidic systems that mimic infection conditions

  • Force measurements to assess membrane mechanical properties

Drawing parallels from studies of the PRD1 infection process, where the internal membrane transforms into a tubular structure that protrudes through a vertex and penetrates the cell envelope for DNA injection , researchers could investigate whether P18 participates in this transformation process, perhaps similarly to how other membrane proteins like P16 interact with vertex structures .

What strategies can be employed to study potential interactions between P18 (XVIII) and the unique portal vertex proteins of PRD1?

Investigating interactions between P18 and portal vertex proteins requires specialized approaches for membrane-associated complexes:

• Genetic Approaches:

  • Construction of PRD1 mutants with tagged versions of P18 and portal proteins

  • Complementation studies with modified proteins

  • Synthetic lethal screens to identify functional relationships

• Biochemical Methods:

  • Chemical cross-linking followed by mass spectrometry

  • Co-immunoprecipitation with membrane-compatible detergents

  • Blue native PAGE for identifying native complexes

• Structural Biology Techniques:

  • Cryo-electron tomography of PRD1 particles, focusing on portal regions

  • Sub-tomogram averaging to enhance resolution

  • Targeted labeling approaches (gold particles, Fab fragments)

• Reconstitution Experiments:

  • In vitro assembly of portal components with and without P18

  • Activity assays for portal functions (e.g., DNA packaging) in the presence/absence of P18

The PRD1 portal vertex contains proteins P6, P9, P20, and P22, with P9 serving as the packaging ATPase . P20 and P22 are small integral membrane proteins that link P6 and P9 to the viral particle . Investigators could explore whether P18 interacts with these proteins or contributes to portal function differently from the known membrane connectors P20 and P22.

How should researchers interpret contradictory results between in vitro and in vivo studies of P18 function?

When faced with contradictions between in vitro and in vivo findings regarding P18 function:

• Systematic Reconciliation Approach:

  • Evaluate methodological differences that might explain discrepancies

  • Consider protein conformation differences between systems

  • Assess lipid composition variations that may affect function

• Quantitative Analysis Framework:

  Parameter	In Vitro Measurement	In Vivo Observation	Potential Reconciliation Strategy
  Binding affinity	Direct measurement with purified components	Inferred from genetic studies	Validate with intermediate complexity systems
  Functional activity	Isolated system measurements	Phenotypic outcomes	Identify missing cofactors or regulators
  Structural details	High-resolution but static	Lower resolution but native	Integrate data using computational modeling

• Complementary Techniques:

  • Develop intermediate complexity systems that bridge the in vitro/in vivo gap

  • Use cellular fractionation to isolate native complexes containing P18

  • Apply genetic approaches that mimic in vitro conditions (e.g., simplified genetic backgrounds)

• Contextual Factors to Consider:

  • Stage of viral life cycle being examined

  • Host cell variations

  • Presence of other viral proteins that modify P18 function

Insights from studies of other PRD1 membrane proteins suggest that context is critical—for example, P16's role in stabilizing spike complexes was only fully understood through the analysis of P16-deficient virions in the context of intact viral particles .

What computational approaches are most useful for predicting P18 structure-function relationships and guiding experimental design?

Computational methods can provide valuable insights for studying P18:

• Structural Prediction Methods:

  • Alpha-helical transmembrane domain prediction tools (TMHMM, HMMTOP)

  • Ab initio modeling with membrane-specific force fields

  • Homology modeling using known structures of similar viral membrane proteins

• Molecular Dynamics Simulations:

  • All-atom simulations in explicit membrane environments

  • Coarse-grained approaches for larger scale dynamics

  • Steered molecular dynamics to assess membrane deformation properties

• Protein-Protein Interaction Prediction:

  • Docking algorithms optimized for membrane proteins

  • Sequence coevolution analysis to identify interacting surfaces

  • Integrative modeling incorporating sparse experimental constraints

• Functional Site Prediction:

  • Conservation analysis across related phages

  • Identification of physicochemical property patterns associated with specific functions

  • Machine learning approaches trained on known viral membrane proteins

• Simulation-Guided Experimental Design:

  • Virtual mutagenesis to identify critical residues

  • Prediction of conformational changes that could be tested experimentally

  • Identification of potential binding sites for probe attachment

Given P18's sequence (MPFGLIVIGIILAIAAYRDTLGELFSIIKDVSKDAKGFGYWVLAAVILGFAASIKPIKEPVNAFMILLMIVLLIRKRGAIDQISNQLRGS) , computational analysis would likely reveal multiple transmembrane regions and potentially specific motifs that could guide targeted experimental studies of its function in the PRD1 viral membrane.

What emerging technologies offer promising applications for studying P18 function in PRD1 biology?

Several cutting-edge technologies show potential for advancing P18 research:

• Cryo-Electron Tomography with Focused Ion Beam Milling:

  • Enables visualization of P18 in intact virions

  • Potential to reveal native organization within the membrane

  • Can capture different states during infection process

• Mass Photometry/Interferometric Scattering Microscopy:

  • Allows label-free detection of protein complexes

  • Can measure heterogeneity in membrane protein assemblies

  • Requires minimal sample amounts

• Nanobody Development:

  • Generation of P18-specific nanobodies as research tools

  • Applications in super-resolution imaging and pull-down assays

  • Potential to trap specific conformational states

• DNA-PAINT Super-Resolution Microscopy:

  • Can achieve sub-10nm resolution of protein organization

  • Allows multiplexed imaging of different viral components

  • Compatible with structural studies of intact virions

• Cell-Free Expression Systems:

  • Rapid prototyping of P18 variants

  • Direct incorporation into artificial membrane systems

  • High-throughput functional screening

• CRISPR-Based Engineering of Host-Phage Systems:

  • Precise genome editing of both host and phage

  • Creation of reporter systems for tracking P18 dynamics

  • Development of selectable markers for evolutionary studies

These technologies could help resolve outstanding questions about P18's role in the context of PRD1's complex membrane biology and infection mechanisms, particularly in relation to the structural and functional differences between the 11 adsorption vertices and the unique portal vertex .

How might comparative studies between P18 and membrane proteins from related phages inform our understanding of viral membrane protein evolution?

Comparative evolutionary analyses offer valuable insights:

• Phylogenetic Analysis Framework:

  • Identification of P18 homologs across the Tectiviridae family and related phages

  • Mapping of conserved domains versus variable regions

  • Correlation of sequence conservation with known functional elements

• Structure-Function Evolutionary Patterns:

  • Analysis of selective pressure on different protein regions

  • Identification of co-evolving residues within P18 or between P18 and interacting partners

  • Assessment of convergent evolution in membrane proteins of unrelated phages

• Experimental Approaches:

  • Functional complementation assays using P18 homologs from related phages

  • Creation of chimeric proteins to map functional domains

  • Reconstruction of ancestral P18 sequences to test evolutionary hypotheses

• Contextual Evolutionary Considerations:

  • Comparison with adenovirus membrane proteins, given PRD1's structural similarities to adenovirus

  • Analysis of adaptation to different host membrane environments

  • Investigation of specialized functions in different viral lineages

This evolutionary perspective would complement the structural studies that have revealed similarities between PRD1 and adenovirus, including the double-barrel trimeric capsid proteins and vertex complexes , potentially identifying conserved principles of viral membrane organization across diverse virus families.

What are the most reliable purification strategies for obtaining high-quality recombinant P18 protein suitable for structural studies?

Purifying membrane proteins like P18 for structural studies requires specialized approaches:

• Optimized Expression Strategies:

  • Use of specialized E. coli strains (C41/C43, Lemo21)

  • Controlled induction conditions (lower temperature, reduced inducer concentration)

  • Addition of specific lipids or chaperones during expression

• Extraction and Solubilization:

  Detergent Class	Examples	Advantages	Limitations
  Mild non-ionic	DDM, LMNG	Gentle, preserves function	Large micelles
  Zwitterionic	LDAO, Fos-choline	Effective solubilization	Can be harsher
  Novel amphipathic agents	SMALPs, amphipols	Maintain native lipid environment	Limited compatibility with some techniques

• Chromatography Sequence:

  • Immobilized metal affinity chromatography (IMAC) utilizing the His-tag

  • Size exclusion chromatography for removing aggregates

  • Optional ion exchange step for removing contaminants

• Quality Control Metrics:

  • Monodispersity assessment by dynamic light scattering

  • Thermal stability assays (differential scanning fluorimetry)

  • Functionality tests in reconstituted systems

• Stabilization for Structural Studies:

  • Screening detergent-lipid combinations

  • Addition of stabilizing ligands if identified

  • Nanobody complexation to rigidify flexible regions

For P18 specifically, the purification would start with the reconstitution protocol mentioned in the product information (centrifugation, reconstitution in deionized water) , followed by more specialized steps depending on the intended structural method.

How can researchers effectively incorporate isotopically labeled P18 protein into structural biology workflows?

Isotopic labeling of P18 enables advanced structural studies:

• Expression Systems for Isotopic Labeling:

  • Minimal media formulations for E. coli expression

  • Carbon sources: [13C]-glucose, [13C]-glycerol

  • Nitrogen sources: [15N]-ammonium chloride

  • Selective labeling approaches for specific amino acids

• Specialized Labeling Strategies:

  • SAIL (Stereo-Array Isotope Labeling) for improved NMR resolution

  • Cell-free expression systems for efficient incorporation of non-canonical amino acids

  • Segmental labeling for focusing on specific regions of P18

• Method-Specific Considerations:

  For NMR Studies:

  • Deuteration strategies to reduce spectral complexity

  • TROSY-based experiments for membrane proteins

  • Specific labeling patterns optimized for membrane protein studies

  For Mass Spectrometry:

  • SILAC approaches for comparative studies

  • Hydrogen-deuterium exchange protocols adapted for membrane proteins

  • Cross-linking MS with isotopically coded cross-linkers

• Integration with Other Structural Data:

  • Combined analysis with cryo-EM data

  • Integration with computational models

  • Validation using complementary biophysical techniques

The experimental design should consider P18's membrane protein nature, its relatively small size (90 amino acids) , and the specific research questions being addressed, such as its potential interactions with other PRD1 structural elements or its role in membrane organization.

How can insights from P18 research contribute to our understanding of viral membrane biogenesis and transformation?

Research on P18 has broader implications:

• Membrane Assembly Models:

  • Insights into how viral membrane proteins coordinate with host lipids

  • Understanding selective incorporation mechanisms for specific lipids

  • Models for membrane curvature induction and maintenance

• Membrane Transformation Mechanisms:

  • Potential role in the tubular membrane transformation observed during PRD1 infection

  • Comparison with other viral systems that undergo membrane remodeling

  • Investigation of protein-lipid interactions that facilitate structural transitions

• Evolutionary Perspectives:

  • Comparison with other internal membrane-containing viruses

  • Analysis of convergent solutions to membrane organization challenges

  • Insights into the origins of viral membrane systems

• Methodological Advances:

  • Development of model systems for studying membrane protein dynamics

  • Improvement of reconstitution methods for complex membrane assemblies

  • New approaches for tracking membrane reorganization events

Understanding P18's role could help explain the unique features of PRD1's membrane system, which differs from host bacterial membranes and undergoes dramatic reorganization during infection to form a tube that penetrates the host cell envelope .

What are the potential applications of recombinant P18 protein beyond basic phage biology research?

Recombinant P18 has potential applications in several areas:

• Biotechnology Applications:

  • Development of novel membrane protein scaffolds

  • Engineering of protein-based membrane penetration systems

  • Creation of biomimetic materials inspired by viral membrane organization

• Methodological Advances:

  • Model system for membrane protein reconstitution protocols

  • Test case for new membrane protein structural determination approaches

  • Platform for developing membrane protein interaction assays

• Therapeutic Development:

  • Understanding fundamental mechanisms of membrane penetration relevant to drug delivery

  • Potential inspiration for designing membrane-active peptides

  • Development of strategies to target bacterial membranes based on phage mechanisms

• Synthetic Biology:

  • Components for engineered virus-like particles

  • Building blocks for artificial membrane systems with programmable properties

  • Elements for creating minimal viral systems

These applications leverage the specialized properties of viral membrane proteins like P18, which have evolved to mediate specific membrane-associated functions and structural transitions that could be repurposed for biotechnological and biomedical applications.

What are the most significant unsolved questions regarding P18 structure and function?

Several critical questions remain unanswered:

• Structural Organization:

  • What is the high-resolution structure of P18 in a membrane environment?

  • How does P18 organize within the PRD1 membrane relative to other membrane proteins?

  • Does P18 undergo conformational changes during the viral life cycle?

• Functional Role:

  • What specific function does P18 serve in the viral membrane?

  • How does P18 contribute to membrane stability or reorganization?

  • Is P18 involved in the membrane tube formation during infection?

• Interactions:

  • Does P18 interact with the vertex proteins (P31, P5, P2) like P16 does ?

  • Is there any functional relationship with the portal proteins (P6, P9, P20, P22) ?

  • Are there specific lipid interactions that are essential for P18 function?

• Evolutionary Context:

  • How conserved is P18 across related phages?

  • Does P18 have functional homologs in other virus families?

  • What evolutionary pressures have shaped P18's sequence and structure?

Addressing these questions will require integrating multiple experimental approaches, from high-resolution structural studies to functional assays in reconstituted systems and intact virions, building upon the existing knowledge of PRD1 biology and membrane protein biophysics.

How might collaborative approaches between structural biology, biophysics, and molecular virology accelerate progress in understanding P18 and related phage membrane proteins?

Interdisciplinary collaboration offers several advantages:

• Integrated Methodological Approaches:

  • Structural biologists providing high-resolution data on P18 conformation

  • Biophysicists characterizing dynamic properties and lipid interactions

  • Virologists establishing functional significance in the viral life cycle

  • Computational biologists developing integrative models that incorporate diverse data types

• Technology Development Synergies:

  • Creation of specialized tools for membrane protein analysis

  • Development of viral membrane-mimetic systems

  • Adaptation of emerging imaging technologies for viral studies

• Knowledge Integration Framework:

  • Systematic data collection across scales (atomic to viral particle)

  • Standardized protocols for comparing results across laboratories

  • Shared resources such as antibodies, expression constructs, and mutant collections

• Accelerated Research Cycles:

  • Hypothesis generation from structural studies informing functional experiments

  • Functional insights guiding targeted structural analyses

  • Iterative refinement of mechanistic models