Recombinant Haemophilus influenzae Uncharacterized protein HI_1413 (HI_1413)

What is Recombinant Haemophilus influenzae Uncharacterized protein HI_1413?

Recombinant Haemophilus influenzae Uncharacterized protein HI_1413 (HI_1413) is a full-length protein comprising 93 amino acids (1-93aa) derived from the Haemophilus influenzae bacterium. In recombinant form, it is typically expressed with an N-terminal histidine tag to facilitate purification and is produced using E. coli expression systems. This protein corresponds to the UniProt accession number P44185 but currently lacks comprehensive functional characterization .

The term "uncharacterized" indicates that while the protein's sequence is known from genomic data, its specific biological function, three-dimensional structure, and role in cellular processes remain to be fully elucidated through experimental approaches.

What expression systems are optimal for producing Recombinant HI_1413?

The optimal expression system for producing Recombinant HI_1413 protein is E. coli, which offers several methodological advantages for this particular protein:

• The protein's relatively small size (93 amino acids) makes it amenable to bacterial expression

• N-terminal His-tagging facilitates subsequent purification via affinity chromatography

• E. coli expression systems generally provide high yield for non-glycosylated bacterial proteins

The expression protocol typically involves:

While mammalian or insect cell expression systems might be considered for complex eukaryotic proteins, they offer no significant advantage for this bacterial protein and would unnecessarily complicate the production process.

What preliminary characterization methods are essential for HI_1413?

Essential preliminary characterization of HI_1413 requires multiple complementary approaches:

• SDS-PAGE and Western Blotting: To confirm protein size (approximately 10-12 kDa plus tag) and purity

• Mass Spectrometry: For precise molecular weight determination and verification of primary sequence

• Circular Dichroism (CD): To assess secondary structure elements

• Dynamic Light Scattering (DLS): To evaluate homogeneity and potential aggregation

• Thermal Shift Assay: To determine protein stability under various buffer conditions

These foundational analyses provide crucial quality control data before proceeding to more sophisticated functional or structural investigations. Researchers should ensure batch-to-batch consistency through these analyses before conducting downstream experiments.

How can bioinformatic approaches predict potential functions of HI_1413?

Bioinformatic prediction of HI_1413 function requires a multi-tool approach:

• Domain Identification: Using NCBI Conserved Domain Search Service (CDD) to identify conserved domains through Reverse Position Specific (RPS)-BLAST against position-specific scoring matrices (PSSMs) . This reveals evolutionary relationships to proteins of known function.

• Homology Analysis: BLASTp comparison against characterized proteins, with particular attention to:

  • Sequence identity ≥35%

  • Query coverage ≥35%

  • E-value <10e-5

• Subcellular Localization Prediction: Tools like PSORT and SignalP identify potential cellular destinations, crucial for narrowing functional hypotheses.

• Structural Prediction: AlphaFold can generate accurate structural models that may reveal functional motifs not apparent from sequence alone .

• Virulence Potential Assessment: Comparison against the Virulence Factor Database (VFDB) to determine if HI_1413 shares features with known bacterial virulence factors .

The integration of these computational approaches generates testable hypotheses about HI_1413 function that guide subsequent experimental design. The absence of significant homologs may suggest novel protein family membership, warranting more extensive experimental characterization.

What structural prediction methods yield the most reliable results for HI_1413?

For structural prediction of HI_1413, a comparative methodology yields the most reliable results:

What experimental approaches are most effective for determining HI_1413 function?

A systematic experimental workflow for determining HI_1413 function should include:

• Protein-Protein Interaction Studies:

  • Pull-down assays using His-tagged HI_1413 as bait

  • Bacterial two-hybrid screening against H. influenzae library

  • Cross-linking mass spectrometry to identify interaction partners

• Gene Knockout/Knockdown Analysis:

  • CRISPR-Cas9 or homologous recombination to create HI_1413 deletion mutants

  • Phenotypic characterization across growth conditions

  • Complementation studies to confirm specificity of observed phenotypes

• Localization Studies:

  • Fluorescent protein fusions or immunofluorescence microscopy

  • Subcellular fractionation followed by Western blotting

• Biochemical Activity Screening:

  • Nucleic acid binding assays

  • Enzymatic activity tests against common substrates

  • Structural stability assessments under various conditions

• Comparative Transcriptomics:

  • RNA-seq analysis comparing wild-type and HI_1413 deletion strains

  • Identification of genes with altered expression patterns

This multi-faceted approach maximizes the likelihood of functional discovery while minimizing false leads that might arise from single-method approaches.

How should experimental contradictions in HI_1413 characterization be resolved?

Resolving experimental contradictions in HI_1413 characterization requires:

• Standardization of Experimental Conditions:

  • Verification of protein quality across experiments (purity, proper folding)

  • Consistent buffer compositions and assay temperatures

  • Standardized strain backgrounds for in vivo experiments

• Orthogonal Method Validation:

  • Confirmation of key findings using methodologically distinct approaches

  • Quantitative assessment of results using statistical analysis

  • Consideration of dose-dependent effects and potential thresholds

• Biological Context Consideration:

  • Growth phase-specific effects

  • Environmental condition dependencies

  • Potential redundancy with functionally related proteins

• Data Integration Through Systems Biology:

  • Network analysis to place contradictory results in broader context

  • Computational modeling to predict conditions where contradictions might be resolved

  • Identification of potential confounding variables

Contradictory results often reveal important biological complexity rather than experimental error. Systematic exploration of the conditions under which different results occur may lead to deeper understanding of context-dependent protein functions.

How does HI_1413 compare to other uncharacterized proteins in Haemophilus influenzae?

Comparative analysis of HI_1413 with other uncharacterized proteins in H. influenzae reveals:

• Sequence Conservation Patterns:

  • HI_1413 (93aa) differs significantly from HI_1543, another uncharacterized protein from H. influenzae with unknown function

  • Conservation analysis across H. influenzae strains indicates phylogenetic distribution patterns that may suggest functional importance

• Domain Architecture:

  • Unlike larger uncharacterized proteins that often contain multiple domains, HI_1413's compact size suggests either a single functional domain or participation in multi-protein complexes

  • Absence of recognizable domains distinguishes it from many other uncharacterized proteins that contain partial matches to known domains

• Expression Patterns:

  • Transcriptomic data analysis can reveal co-expression patterns with genes of known function

  • Differential expression under stress conditions compared to other uncharacterized proteins

• Structural Predictions:

  • AlphaFold2 predictions may reveal structural similarity to proteins of known function despite low sequence identity

  • Comparison of predicted binding sites across multiple uncharacterized proteins

This comparative approach places HI_1413 in context among the approximately 300-400 uncharacterized proteins in the H. influenzae proteome, potentially identifying functional clusters.

What conservation patterns of HI_1413 exist across bacterial species?

Cross-species conservation analysis of HI_1413 provides crucial evolutionary context:

• Phylogenetic Distribution:

  • Conservation primarily within Pasteurellaceae family

  • Potential distant homologs in other Gammaproteobacteria

  • Absence in Gram-positive bacteria would suggest function specific to Gram-negative cellular architecture

• Synteny Analysis:

  • Conservation of genomic context across species

  • Co-evolution with functionally related genes

  • Presence in essential gene clusters versus accessory genome regions

• Selection Pressure Metrics:

  • dN/dS ratios indicating intensity of evolutionary constraints

  • Identification of highly conserved residues as potential functional hotspots

  • Detection of recent evolutionary adaptations

• Deep Green Analysis Approach:

  • While plants like Arabidopsis thaliana and Setaria viridis have been used to identify conserved uncharacterized proteins across the green lineage , a similar approach could identify deeply conserved bacterial proteins

  • Such conservation would suggest fundamental biological roles

Proteins conserved across diverse bacterial species typically serve fundamental cellular functions, while narrowly distributed proteins often relate to specialized adaptations or host-pathogen interactions.

What specialized techniques are recommended for detailed structural analysis of HI_1413?

For comprehensive structural characterization of HI_1413, researchers should consider:

• X-ray Crystallography:

  • Optimization of crystallization conditions through sparse matrix screening

  • Use of fusion proteins (e.g., T4 lysozyme) to aid crystallization

  • Molecular replacement using AlphaFold2 models as search templates

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Particularly suitable for HI_1413 due to its small size (93aa)

  • 2D and 3D heteronuclear experiments for backbone and side-chain assignments

  • Relaxation measurements to identify flexible regions

• Cryo-Electron Microscopy:

  • Most beneficial if HI_1413 forms larger complexes with binding partners

  • Single-particle analysis for structural determination

  • Tomography for in situ structural arrangements

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Identification of solvent-accessible regions

  • Analysis of structural dynamics

  • Mapping of interaction interfaces

• Small-Angle X-ray Scattering (SAXS):

  • Low-resolution envelope determination

  • Assessment of oligomeric state in solution

  • Validation of computational models

The integration of multiple structural techniques provides complementary information, addressing the limitations of individual methods.

How can AlphaFold predictions enhance experimental structural studies of HI_1413?

AlphaFold predictions substantially enhance experimental structural studies of HI_1413 through:

• Crystallographic Phasing:

  • Molecular replacement using AlphaFold models as search templates

  • Phase improvement in cases of low-resolution diffraction data

  • Guide for construct design to remove disordered regions that hinder crystallization

• NMR Spectral Assignment:

  • Prediction-guided assignment strategies for backbone resonances

  • Identification of secondary structure elements to facilitate assignment

  • Constraint generation for structure calculation

• Experimental Design Guidance:

  • Identification of potential binding sites for mutagenesis studies

  • Design of truncation constructs based on predicted domain boundaries

  • Selection of optimal labeling sites for biophysical studies

• Model Validation:

  • pLDDT scores provide confidence metrics for different regions

  • Comparison between predicted and experimental structures highlights regions of dynamic behavior

  • Identification of potential artifacts in experimental structures

How might HI_1413 contribute to Haemophilus influenzae pathogenicity?

Assessment of HI_1413's potential role in pathogenicity requires:

• Virulence Association Analysis:

  • Comparison against the Virulence Factor Database (VFDB) to identify similarities with known virulence factors

  • Evaluation of genomic context for proximity to pathogenicity islands

  • Examination of expression patterns during infection models

• Host Interaction Potential:

  • Homology analysis against human proteome to assess potential cross-reactivity

  • Prediction of surface exposure and accessibility to host immune system

  • Screening for interactions with host receptors or immune components

• Differential Expression Analysis:

  • Comparison of expression levels between virulent and avirulent strains

  • Transcriptomic profiling during different infection stages

  • Response to host-mimicking stressors (oxidative stress, nutrient limitation)

• Functional Context Assessment:

  • Association with membrane structures essential for host colonization

  • Potential role in biofilm formation or antibiotic resistance

  • Involvement in metabolic pathways activated during infection

While many uncharacterized proteins ultimately prove essential for bacterial physiology rather than direct virulence, understanding their roles can still identify valuable therapeutic targets, particularly if they perform functions essential for pathogen survival during infection.

What potential exists for HI_1413 as a therapeutic target or diagnostic marker?

Evaluation of HI_1413 as a therapeutic target or diagnostic marker involves:

• Target Validation Criteria:

  • Essentiality assessment through gene knockout studies

  • Conservation analysis across clinically relevant strains

  • Absence of human homologs to minimize off-target effects

  • Accessibility to small molecules or biologics

• Diagnostic Potential Assessment:

  • Antibody development against recombinant HI_1413

  • Evaluation of expression levels during infection

  • Specificity testing against related bacterial species

  • Detection sensitivity in clinical samples

• Structure-Based Drug Design Opportunities:

  • Identification of druggable pockets in the predicted structure

  • Virtual screening of compound libraries against structural models

  • Fragment-based drug discovery approaches

• Immunogenic Potential Evaluation:

  • Prediction of B-cell and T-cell epitopes

  • Assessment of surface accessibility of immunogenic regions

  • Cross-reactivity analysis with human proteins

  • Conservation of epitopes across clinically relevant strains

Uncharacterized proteins like HI_1413 represent untapped opportunities for novel therapeutic approaches, particularly if they perform essential functions with structural features distinct from human proteins.