Recombinant Haemophilus influenzae Uncharacterized protein HI_0974.1 (HI_0974.1)

What is known about the structural characteristics of HI_0974.1?

HI_0974.1 is a small protein consisting of 85 amino acids with the sequence: MTLKQRYQQAGKEASWALSLSILYVIGWCLCAYLPKETQGPIGFPLWFELSCIYLPILFI VIGHWIIKIIFQDISLEINDQGNQK . The protein's small size suggests it may function as a regulatory protein or membrane component, particularly given the presence of hydrophobic regions that suggest possible membrane association. While no crystal structure is currently available in the Protein Data Bank, computational modeling using homology-based approaches may provide preliminary structural insights.

What expression systems are recommended for studying HI_0974.1?

E. coli expression systems have been successfully used to produce recombinant HI_0974.1, typically with an N-terminal His-tag to facilitate purification . For optimal expression, consider using BL21(DE3) strains with IPTG induction under standard conditions (0.5-1.0 mM IPTG, 37°C for 4-6 hours). Given the protein's small size, expression yields are typically favorable, though membrane-associated properties may necessitate optimization of solubilization conditions using mild detergents such as n-dodecyl β-D-maltoside (DDM) or Triton X-100.

What purification methods are effective for recombinant HI_0974.1?

Since recombinant HI_0974.1 is typically produced with a His-tag, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin represents the primary purification method . A recommended protocol includes:

• Cell lysis using sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole

• Binding to Ni-NTA resin with gentle agitation (4°C, 1 hour)

• Sequential washing with increasing imidazole concentrations (20-50 mM)

• Elution with 250-300 mM imidazole

• Size exclusion chromatography as a polishing step

This approach typically yields protein with >90% purity suitable for functional studies .

How should purified HI_0974.1 be stored for maximum stability?

For optimal stability, store HI_0974.1 as a lyophilized powder at -20°C to -80°C for long-term storage . For working aliquots, reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL and add glycerol to a final concentration of 50% before storing at -20°C . Avoid repeated freeze-thaw cycles as they can compromise protein integrity. Working aliquots can be maintained at 4°C for up to one week . When reconstituting, a gentle centrifugation is recommended to bring contents to the bottom of the vial.

What experimental approaches can determine the function of uncharacterized protein HI_0974.1?

To systematically characterize HI_0974.1 function, implement a multi-faceted strategy:

• Comparative genomics analysis: Identify potential orthologs across bacterial species, particularly focusing on conserved genomic context that might suggest functional relationships .

• Transcriptomic profiling: Compare expression patterns across various growth conditions and stress responses to identify conditions where HI_0974.1 is significantly up- or down-regulated.

• Gene knockout studies: Create deletion mutants of HI_0974.1 in H. influenzae and assess phenotypic changes in growth, virulence, and stress responses. RNAi screening approaches have proven effective for similar uncharacterized genes in other organisms .

• Protein-protein interaction studies: Employ pull-down assays, bacterial two-hybrid screens, or proximity labeling techniques to identify interaction partners that may suggest functional pathways.

• Structural studies: If crystallization proves challenging, consider NMR spectroscopy for this relatively small protein to gain structural insights that may inform function.

How can researchers design effective experimental controls when studying HI_0974.1?

Table 1: Recommended Controls for HI_0974.1 Experiments

When designing experiments, consider between-subjects or within-subjects approaches depending on your specific question, and ensure proper randomization to minimize bias . Document all variables carefully, distinguishing between independent variables (e.g., protein concentration), dependent variables (e.g., binding affinity), and potential confounding variables.

What bioinformatic approaches can predict functional domains in HI_0974.1?

For comprehensive in silico functional prediction, employ a hierarchical bioinformatic workflow:

• Sequence-based analysis:

  • Profile hidden Markov model searches using HMMER against Pfam and other domain databases

  • Position-specific scoring matrix searches using PSI-BLAST

  • Identification of short linear motifs using ELM

• Structural prediction:

  • Generate 3D models using AlphaFold2 or RoseTTAFold

  • Analyze fold recognition using protein threading (PHYRE2)

  • Identify potential binding pockets using SiteMap or CASTp

• Evolutionary analysis:

  • Calculate conservation scores across orthologs using ConSurf

  • Perform evolutionary trace analysis to identify functionally important residues

  • Assess co-evolution patterns suggesting interaction interfaces

• Genomic context:

  • Analyze operonic structure and gene neighborhood

  • Identify phylogenetic profiles across bacterial species

  • Examine fusion events with characterized proteins

This systematic approach has successfully identified functions for previously uncharacterized bacterial proteins and can be adapted specifically for HI_0974.1.

How can researchers investigate potential roles of HI_0974.1 in H. influenzae pathogenesis?

To assess pathogenic relevance, implement the following experimental strategy:

• Infection models:

  • Develop wild-type and HI_0974.1 knockout strains for comparative studies in appropriate infection models

  • Monitor bacterial survival, proliferation, and host response metrics

• Immune interaction studies:

  • Assess host immune recognition of purified HI_0974.1

  • Measure cytokine responses in immune cells exposed to the protein

  • Investigate interaction with pattern recognition receptors

• Comparative virulence analysis:

  • Compare growth kinetics in host-mimicking conditions between wild-type and knockout strains

  • Assess biofilm formation capabilities

  • Evaluate antibiotic susceptibility profiles

• Transcriptional response:

  • Perform RNA-seq on host cells exposed to wild-type versus knockout strains

  • Identify differentially regulated host defense genes

• Vaccine potential assessment:

  • Evaluate immunogenicity of recombinant HI_0974.1

  • Assess protective efficacy in appropriate animal models

What approaches can resolve data contradictions in HI_0974.1 research?

When facing contradictory research outcomes, implement this systematic resolution framework:

• Methodological reconciliation:

  • Create a comprehensive data table comparing experimental conditions across studies

  • Identify key variables that differ between contradictory results

  • Design controlled experiments specifically testing these variables

• Statistical reanalysis:

  • Perform meta-analysis when sufficient studies exist

  • Consider Bayesian approaches to integrate prior knowledge with new data

  • Evaluate effect sizes rather than simply p-values

• Technical validation:

  • Independently reproduce key findings using multiple methods

  • Validate results using orthogonal techniques

  • Consider blind experimental design to minimize bias

• Biological context integration:

  • Examine if contradictions reflect genuine biological variability

  • Consider strain differences, growth conditions, or post-translational modifications

  • Develop testable models that could explain apparent contradictions

This systematic approach allows researchers to transform contradictions into new hypothesis-generating opportunities.

How can researchers determine if HI_0974.1 interacts with host proteins during infection?

To systematically investigate host-pathogen protein interactions involving HI_0974.1:

• In vitro binding assays:

  • Express and purify His-tagged HI_0974.1

  • Perform pull-down assays using host cell lysates

  • Identify binding partners via mass spectrometry

  • Confirm direct interactions using surface plasmon resonance or microscale thermophoresis

• Yeast two-hybrid screening:

  • Use HI_0974.1 as bait against human cDNA libraries

  • Validate positive interactions with additional biochemical methods

• In vivo approaches:

  • Employ proximity labeling techniques (BioID or APEX) with HI_0974.1 expressed during infection

  • Perform immunoprecipitation of cross-linked complexes during active infection

  • Use fluorescence resonance energy transfer (FRET) to visualize interactions in real-time

• Computational prediction:

  • Apply host-pathogen interaction prediction algorithms

  • Dock predicted structural models against human protein structures

  • Identify potential binding motifs shared with known virulence factors

This multi-faceted approach maximizes the likelihood of identifying biologically significant interactions that could inform therapeutic targeting.

What does the hydrophobicity profile of HI_0974.1 suggest about its cellular localization?

Analysis of the HI_0974.1 sequence (MTLKQRYQQAGKEASWALSLSILYVIGWCLCAYLPKETQGPIGFPLWFELSCIYLPILFI VIGHWIIKIIFQDISLEINDQGNQK) reveals:

• Transmembrane domain prediction:

  • The central region (approximately residues 15-40) contains a stretch of hydrophobic amino acids consistent with a potential transmembrane domain

  • The sequence contains multiple leucine, isoleucine, and valine residues arranged in patterns typical of membrane-spanning regions

• Amphipathic helices:

  • The N-terminal region (residues 1-14) contains both charged (K, R) and hydrophobic residues, consistent with an amphipathic helix

  • Similarly, the C-terminal region shows mixed hydrophobicity

• Experimental validation approaches:

  • Subcellular fractionation of H. influenzae followed by Western blotting

  • Fluorescent protein fusions to visualize localization

  • Membrane extraction assays using increasing detergent concentrations

  • Protease accessibility assays to determine topology

What are the optimal conditions for reconstituting lyophilized HI_0974.1 protein?

To ensure maximum recovery of functional protein from lyophilized HI_0974.1 preparations, follow this optimized protocol:

• Initial preparation:

  • Allow the vial to equilibrate to room temperature before opening

  • Briefly centrifuge to collect the lyophilized powder at the bottom of the vial

• Reconstitution buffer selection:

  • For general applications: Tris/PBS-based buffer, pH 8.0 containing 6% trehalose

  • For membrane protein studies: Consider adding 0.1% DDM or other mild detergent

  • For structural studies: 20 mM sodium phosphate, 150 mM NaCl, pH 7.4

• Reconstitution procedure:

  • Add buffer slowly down the side of the vial

  • Aim for a final concentration of 0.1-1.0 mg/mL

  • Allow to stand for 10-15 minutes before gentle mixing

  • Avoid vortexing; instead, use gentle inversion or rotation

• Storage preparation:

  • Add glycerol to a final concentration of 50%

  • Prepare single-use aliquots to avoid freeze-thaw cycles

  • Flash freeze in liquid nitrogen before transferring to -80°C storage

Following these guidelines will help maintain protein integrity for downstream applications, particularly important for functional studies of this uncharacterized protein.

How can researchers troubleshoot low yields when expressing recombinant HI_0974.1?

When facing challenges with HI_0974.1 expression, implement this systematic troubleshooting approach:

Table 2: Troubleshooting Matrix for HI_0974.1 Expression

Begin by optimizing expression conditions through a small-scale expression matrix varying temperature (18°C, 25°C, 37°C), inducer concentration (0.1, 0.5, 1.0 mM IPTG), and induction time (4, 8, 16 hours). Monitor expression through time-course sampling and analysis by SDS-PAGE.

What crystallization strategies are recommended for structural studies of HI_0974.1?

For crystallizing this challenging membrane-associated uncharacterized protein:

• Protein preparation optimization:

  • Achieve highest possible purity (>95% by SDS-PAGE)

  • Remove His-tag if possible using precision protease

  • Perform thermal shift assays to identify stabilizing buffer conditions

  • Consider limited proteolysis to identify stable domains

• Crystallization screening approach:

  • Begin with sparse matrix screens designed for membrane proteins

  • Implement both vapor diffusion and lipidic cubic phase methods

  • Screen in presence of various detergents (DDM, LDAO, C12E8)

  • Include potential ligands or interacting peptides to stabilize structure

• Optimization strategies:

  • For promising conditions, create focused grid screens varying precipitant concentration, pH, and additives

  • Try seeding from microcrystals

  • Test different protein:reservoir ratios

  • Consider crystallization at different temperatures (4°C, 18°C)

• Alternative approaches:

  • If crystallization proves challenging, consider:

    • NMR for solution structure (feasible for 85-residue protein)

    • Cryo-EM with suitable scaffolds or nanodiscs

    • Fusion with crystallization chaperones (T4 lysozyme, BRIL)

This multi-faceted approach maximizes the likelihood of obtaining structural data for this previously uncharacterized protein.

What are the most promising research directions for understanding HI_0974.1 function?

Based on current knowledge, the following research avenues hold particular promise:

• Comparative genomics expansion:

  • Systematic analysis of HI_0974.1 orthologs across bacterial species can identify evolutionarily conserved features suggesting functional importance

  • Genomic context analysis may reveal consistent co-occurrence with genes of known function

• Structural biology integration:

  • Obtaining high-resolution structural data will significantly accelerate functional understanding

  • Structure-guided mutagenesis targeting predicted functional sites can validate hypotheses

• Host-pathogen interaction focus:

  • Given Haemophilus influenzae's pathogenic nature, investigating HI_0974.1's potential role in virulence represents a high-value research direction

  • Targeting studies to infection-relevant conditions may reveal context-specific functions

• Systems biology approaches:

  • Integration of transcriptomic, proteomic, and metabolomic data can place HI_0974.1 within broader cellular networks

  • Genome-wide screens for synthetic lethality or genetic interactions can reveal functional relationships

These approaches, pursued concurrently, will most efficiently advance understanding of this uncharacterized protein, potentially revealing new biological mechanisms and therapeutic targets.