Recombinant Haemophilus influenzae Uncharacterized protein HI_1016 (HI_1016)

How does HI_1016 relate to other uncharacterized protein families in bacterial systems?

While HI_1016 itself is an uncharacterized protein from Haemophilus influenzae, it may share structural or functional similarities with other uncharacterized protein families. For comparison, the Uncharacterized Protein Family 0016 (UPF0016) represents another group of poorly studied membrane proteins that are well-conserved throughout evolution . Members of UPF0016 contain conserved motifs and have been identified as transporters of cations, particularly Mn²⁺, with additional reported functions in Ca²⁺ and/or H⁺ transport .

To determine potential relationships between HI_1016 and other protein families, researchers should conduct computational analyses including:

• Multiple sequence alignments to identify conserved domains

• Phylogenetic analyses to determine evolutionary relationships

• Structural prediction to identify potential functional motifs

• Comparative genomics across different bacterial species

This approach may reveal whether HI_1016 belongs to a larger family of functionally related proteins or represents a unique protein specific to Haemophilus influenzae.

What are the optimal conditions for the expression of recombinant HI_1016 protein in E. coli systems?

The expression of recombinant HI_1016 in E. coli should be optimized using a systematic Design of Experiments (DoE) approach rather than the inefficient one-factor-at-a-time method. This approach allows for the assessment of multiple parameters simultaneously while minimizing experimental runs .

Key parameters to optimize include:

• Expression vector selection: pET series vectors with T7 promoter systems are commonly used for high-level expression

• E. coli strain selection: BL21(DE3), Rosetta, or Origami strains depending on codon usage and disulfide bond formation needs

• Induction conditions:

  • IPTG concentration (typically 0.1-1.0 mM)

  • Induction temperature (16-37°C, with lower temperatures often favoring proper folding)

  • Induction duration (3-24 hours)

• Media composition: LB, TB, or minimal media with appropriate antibiotics

• Cell density at induction: Typically at OD₆₀₀ of 0.6-0.8

A fractional factorial design can be implemented to screen these factors, followed by response surface methodology for fine-tuning the most significant parameters . Software packages are available to facilitate DoE approach selection, experiment design, and results analysis, ultimately leading to optimized expression conditions with reduced time and resource investment.

What purification strategies are most effective for obtaining high-purity HI_1016 protein for structural and functional studies?

For His-tagged recombinant HI_1016, a multi-step purification strategy is recommended:

• Initial capture using IMAC (Immobilized Metal Affinity Chromatography):

  • Ni-NTA or Co-NTA columns with imidazole gradient elution

  • Typical binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Elution buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250-500 mM imidazole

• Secondary purification:

  • Size exclusion chromatography to remove aggregates and obtain monodisperse protein

  • Ion exchange chromatography if charge-based separation is beneficial

• Quality control assessments:

  • SDS-PAGE to confirm >90% purity

  • Western blot using anti-His antibodies

  • Mass spectrometry to verify protein identity and integrity

  • Dynamic light scattering to assess monodispersity

• Buffer optimization for stability:

  • The protein is typically stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0

  • Addition of 5-50% glycerol (final concentration) is recommended for long-term storage at -20°C/-80°C

  • Aliquoting is necessary to avoid repeated freeze-thaw cycles which can compromise protein stability

The purified protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL for experimental use .

What computational and experimental methods can be employed to predict the function of HI_1016?

A comprehensive approach combining computational and experimental methods is recommended:

Computational Methods:

• Sequence-based analysis:

  • Homology searches using BLAST, HHpred, or PSI-BLAST

  • Motif identification using PROSITE, Pfam, or SMART

  • Secondary structure prediction using PSIPRED or JPred

  • 3D structure prediction using AlphaFold2 or RoseTTAFold

• Genomic context analysis:

  • Examining neighboring genes that may be functionally related

  • Identifying potential operons that include HI_1016

  • Comparative genomics across different Haemophilus strains

• Evolutionary analysis:

  • Phylogenetic profiling to identify co-evolved proteins

  • Identification of conserved residues that may be functionally important

Experimental Methods:

• Protein-protein interaction studies:

  • Pull-down assays using the His-tagged protein

  • Yeast two-hybrid screening

  • Cross-linking coupled with mass spectrometry

• Phenotypic analysis:

  • Generation of knockout/knockdown strains

  • Complementation studies

  • Growth under various stress conditions

• Biochemical characterization:

  • Enzymatic activity assays based on computational predictions

  • Binding assays with potential substrates or partners

  • Structural studies (X-ray crystallography, cryo-EM, or NMR)

This integrated approach has been successfully applied to annotate functions of 296 hypothetical proteins from Haemophilus influenzae with high confidence and an additional 124 proteins with lower confidence .

How can researchers effectively design experiments to determine if HI_1016 is involved in cation transport similar to other uncharacterized protein families?

If investigating potential cation transport function of HI_1016 similar to UPF0016 family members , the following experimental design is recommended:

• Membrane localization confirmation:

  • Subcellular fractionation followed by western blotting

  • Fluorescence microscopy with tagged versions of HI_1016

  • Protease protection assays to determine topology

• Transport activity assessment:

  • Reconstitution into liposomes with fluorescent ion indicators

  • Radioisotope flux assays (⁴⁵Ca²⁺, ⁵⁴Mn²⁺)

  • Patch-clamp electrophysiology in heterologous expression systems

• Ion specificity determination:

  • Competitive inhibition assays with various cations

  • Mutagenesis of predicted ion-binding residues

  • ITC (Isothermal Titration Calorimetry) to measure binding affinities

• Physiological relevance testing:

  • Growth assays under ion limitation or excess

  • Metal sensitivity/resistance phenotypes in knockout strains

  • Complementation with known transporters

Experimental setup for cation transport assay in liposomes:

Time-course measurements and dose-response curves should be generated for comprehensive characterization of transport kinetics.

How can researchers experimentally determine the structural features of HI_1016 that might contribute to its function?

To elucidate structure-function relationships in HI_1016, a multi-faceted structural biology approach is recommended:

• High-resolution structure determination:

  • X-ray crystallography: Requires screening of crystallization conditions using commercial kits with varying precipitants, buffers, and additives

  • Cryo-EM: Particularly useful if HI_1016 forms larger complexes or is membrane-associated

  • NMR spectroscopy: Suitable for analyzing dynamics if protein size permits (<25 kDa for routine studies)

• Structural analysis with computational tools:

  • Identification of potential active sites or binding pockets

  • Electrostatic surface mapping to identify charged regions

  • Molecular dynamics simulations to identify flexible regions

• Site-directed mutagenesis studies:

  • Alanine scanning of conserved residues

  • Conservative and non-conservative mutations of predicted functional residues

  • Creation of truncation variants to identify minimal functional domains

• Biophysical characterization:

  • Circular dichroism to assess secondary structure content

  • Thermal shift assays to identify stabilizing conditions or ligands

  • Small-angle X-ray scattering (SAXS) for solution structure

The systematic application of these methods, coupled with functional assays, can establish correlations between specific structural elements and functional properties of HI_1016.

What are the challenges in crystallizing HI_1016 for structural studies, and how can these be addressed?

Crystallization of recombinant proteins like HI_1016 presents several challenges that can be addressed through systematic optimization:

Common challenges and solutions:

• Protein heterogeneity:

  • Ensure monodispersity through rigorous size exclusion chromatography

  • Consider removal of flexible regions identified through limited proteolysis

  • Assess post-translational modifications that may cause heterogeneity

• Buffer optimization:

  • Screen various buffers, pH conditions, and salt concentrations

  • Include stabilizing agents like glycerol, trehalose, or specific ions

  • Consider the addition of reducing agents to prevent oxidation of cysteine residues

• Crystallization screening:

  • Implement sparse matrix screening with commercial kits

  • Use nanoliter-scale crystallization robots to maximize coverage of conditions

  • Consider both vapor diffusion and microbatch methods

• Crystal optimization:

  • Fine-tune promising conditions through grid screens

  • Employ seeding techniques to improve crystal quality

  • Try additive screens to enhance crystal packing

• Alternative approaches:

  • Fusion partners like T4 lysozyme or BRIL to aid crystallization

  • Surface entropy reduction through mutation of surface residues

  • Co-crystallization with antibodies or natural binding partners

A DoE approach can be particularly valuable for crystallization optimization, allowing for the systematic exploration of multiple parameters simultaneously . This methodology has been demonstrated to significantly improve efficiency in finding optimal crystallization conditions compared to traditional one-factor-at-a-time approaches.

How can researchers design experiments to investigate the potential role of HI_1016 in H. influenzae pathogenesis?

To investigate the potential role of HI_1016 in pathogenesis, a comprehensive experimental approach is needed:

• Gene knockout and complementation studies:

  • Create precise deletion mutants using homologous recombination

  • Perform complementation with wild-type and mutated versions

  • Assess virulence phenotypes in appropriate infection models

• Transcriptional analysis:

  • RNA-seq under infection-relevant conditions

  • qRT-PCR to validate expression patterns

  • Promoter activity analysis using reporter constructs

• Host-pathogen interaction studies:

  • Adhesion and invasion assays with human cell lines

  • Immune response assessment (cytokine production, neutrophil recruitment)

  • Bacterial survival in serum, phagocytes, or biofilm formation

• In vivo infection models:

  • Mouse models of bacteremia, pneumonia, or meningitis

  • Competitive index assays (wild-type vs. mutant)

  • Bacterial load determination in various tissues

• Comparative analysis across clinical isolates:

  • Sequence variation analysis of HI_1016 in clinical strains

  • Correlation of variants with virulence or antibiotic resistance

  • Expression levels in antibiotic-resistant versus sensitive strains

This systematic approach can help determine whether HI_1016 contributes to the pathogen's ability to cause bacteremia, pneumonia, or acute bacterial meningitis, which are common manifestations of H. influenzae infection .

What approaches can be used to study the potential interactions between HI_1016 and other proteins in the H. influenzae proteome?

To comprehensively map protein-protein interactions involving HI_1016:

• Affinity-based methods:

  • Co-immunoprecipitation with anti-His antibodies for tagged HI_1016

  • Pull-down assays using immobilized HI_1016 as bait

  • Proximity labeling techniques like BioID or APEX2

• Genetic-based methods:

  • Bacterial two-hybrid systems

  • Suppressor screens to identify genetic interactions

  • Synthetic lethality/fitness studies with other gene knockouts

• High-throughput screens:

  • Protein microarrays using the H. influenzae proteome

  • Mass spectrometry-based interactomics

  • Crosslinking coupled with mass spectrometry (XL-MS)

• Computational predictions:

  • Network analysis based on gene co-expression

  • Structural docking with potential partner proteins

  • Text mining of published literature for potential interactions

• Validation and characterization:

  • Surface plasmon resonance to measure binding kinetics

  • Fluorescence resonance energy transfer (FRET) to confirm interactions in vivo

  • Co-crystallization of HI_1016 with interaction partners

The results from these studies can be integrated into a protein interaction network to place HI_1016 in a functional context within the H. influenzae proteome, potentially revealing its role in specific cellular processes or pathways.

How does the sequence and predicted function of HI_1016 compare across different strains of H. influenzae and related bacterial species?

Understanding the evolutionary context of HI_1016 requires comparative analysis across different strains and related species:

• Sequence conservation analysis:

  • Multiple sequence alignment of HI_1016 homologs from various H. influenzae strains

  • Identification of conserved domains and residues

  • Calculation of selective pressure (dN/dS ratio) to identify regions under selection

• Phylogenetic analysis:

  • Construction of phylogenetic trees to visualize evolutionary relationships

  • Reconciliation with species trees to identify potential horizontal gene transfer events

  • Analysis of gene presence/absence patterns across the Pasteurellaceae family

• Synteny analysis:

  • Examination of gene neighborhood conservation

  • Identification of conserved operons containing HI_1016 homologs

  • Analysis of co-evolution patterns with functionally related genes

• Structural comparison:

  • Homology modeling of HI_1016 from different strains

  • Identification of structural variations that might affect function

  • Analysis of surface properties and potential interaction interfaces

This comparative approach can reveal whether HI_1016 represents a core gene in H. influenzae with a conserved function or shows strain-specific adaptations that might correlate with different pathogenic potentials or ecological niches.

What experimental designs can help determine if HI_1016 contributes to antibiotic resistance in H. influenzae?

Given the increasing concern about multi-drug resistant H. influenzae strains , investigating HI_1016's potential role in antibiotic resistance requires:

• Comparative expression analysis:

  • qRT-PCR comparing HI_1016 expression in sensitive vs. resistant strains

  • RNA-seq under antibiotic stress conditions

  • Promoter activity analysis using reporter constructs

• Genetic manipulation studies:

  • Overexpression of HI_1016 in sensitive strains to assess MIC changes

  • Knockout/knockdown in resistant strains to determine contribution

  • Complementation with variants found in resistant clinical isolates

• Antibiotic susceptibility testing:

  • Determination of MICs for various antibiotics in wild-type and mutant strains

  • Time-kill kinetics to assess rate of antibiotic action

  • Biofilm formation and antibiotic penetration studies

• Mechanistic investigations:

  • Antibiotic accumulation assays to assess potential efflux function

  • Membrane permeability studies using fluorescent dyes

  • Direct binding assays between HI_1016 and antibiotics

Experimental design for antibiotic susceptibility testing:

Testing should include multiple antibiotic classes to identify specific or broad-spectrum effects on resistance profiles.