Recombinant Haemophilus influenzae Uncharacterized protein HI_1736 (HI_1736)

What is Haemophilus influenzae Uncharacterized protein HI_1736?

Haemophilus influenzae Uncharacterized protein HI_1736 is a 77-amino acid protein encoded by the HI_1736 gene from Haemophilus influenzae, identified with UniProt ID P44300. The protein is classified as "uncharacterized" because its specific biological function has not yet been fully determined through experimental validation. Commercial recombinant versions typically feature an N-terminal His-tag to facilitate purification and experimental detection. The protein is generally expressed in E. coli expression systems for research purposes, with the full-length protein (amino acids 1-77) being the standard form available for laboratory investigations .

The uncharacterized status of this protein makes it an interesting target for fundamental research into bacterial protein function and potentially for understanding Haemophilus influenzae pathogenicity mechanisms. Haemophilus influenzae is a significant human pathogen that can cause various infections, with certain serotypes like Hia associated with serious clinical outcomes including intracranial infections .

What structural prediction methods are most appropriate for analyzing HI_1736?

Given the challenges of experimentally determining the structure of small membrane proteins like HI_1736, computational prediction methods provide valuable initial insights. Researchers should employ a multi-faceted approach to structural prediction:

• Transmembrane topology prediction: Tools such as TMHMM, Phobius, or TOPCONS should be used to identify potential membrane-spanning regions in the HI_1736 sequence. Based on the high hydrophobicity profile observed in the amino acid sequence, multiple transmembrane helices are likely present .

• Secondary structure prediction: Methods such as PSIPRED or JPred can estimate the proportion of alpha-helical, beta-strand, and coil regions. For membrane proteins like HI_1736, alpha-helical predictions predominate in transmembrane regions.

• Homology modeling: Although challenging if close homologs with known structures are unavailable, tools like SWISS-MODEL or I-TASSER may identify distant structural relatives to generate approximate models.

• Ab initio modeling: For novel folds with limited homology to known structures, Rosetta membrane protocol or AlphaFold can generate plausible structural models based on physical principles and statistical potentials.

• Molecular dynamics simulations: Once preliminary models are generated, embedding the protein in a simulated lipid bilayer can assess structural stability and dynamic behavior.

For experimental validation of these predictions, techniques such as circular dichroism spectroscopy can determine secondary structure content, while more detailed structural analysis would require NMR spectroscopy (challenging for membrane proteins) or X-ray crystallography following successful crystallization trials.

What expression systems and purification strategies are optimal for obtaining functional HI_1736 protein?

Obtaining functional HI_1736 protein requires careful consideration of expression systems and purification strategies appropriate for membrane-associated bacterial proteins:

Expression Systems Comparison:

Purification Protocol for Functional HI_1736:

• Membrane Extraction: After expression in E. coli, cells should be lysed (sonication or French press), followed by differential centrifugation to isolate membrane fractions. Solubilization requires careful detergent selection - mild detergents like DDM (n-Dodecyl β-D-maltoside) or LMNG (Lauryl Maltose Neopentyl Glycol) are recommended to maintain native structure.

• Affinity Chromatography: The His-tagged protein can be purified using Ni-NTA affinity chromatography with detergent present in all buffers. A step gradient elution with imidazole (50-300mM) typically yields good separation .

• Secondary Purification: Size exclusion chromatography in buffer containing detergent above critical micelle concentration further improves purity and removes aggregates.

• Quality Control: SDS-PAGE should confirm purity >90% , while dynamic light scattering can assess monodispersity. Circular dichroism spectroscopy can verify proper secondary structure formation.

• Functional Verification: Activity assays specific to the hypothesized function should be performed to confirm that the purified protein maintains its native properties.

For reconstitution into proteoliposomes, a gradual detergent removal approach using Bio-Beads or dialysis is recommended to incorporate the protein into a lipid environment that mimics its native membrane.

What biochemical and biophysical methods are most effective for characterizing the function of previously uncharacterized proteins like HI_1736?

A systematic, multi-technique approach is essential for unraveling the function of uncharacterized proteins like HI_1736:

Stage 1: Computational Analysis and Hypothesis Generation

• Genomic context analysis: Examine neighboring genes and operon structure in Haemophilus influenzae to identify functional relationships.

• Phylogenetic profiling: Identify co-occurrence patterns with proteins of known function across bacterial species.

• Structural prediction: Use AlphaFold or similar tools to predict binding pockets or functional domains.

• Expression correlation analysis: Analyze transcriptomic data to identify co-expressed genes during infection or stress conditions.

Stage 2: Biochemical Characterization

• Protein-protein interaction studies:

  • Pull-down assays using His-tagged HI_1736

  • Bacterial two-hybrid screening

  • Chemical cross-linking followed by mass spectrometry

  • Surface plasmon resonance for quantitative binding parameters

• Lipid interaction analysis:

  • Lipid overlay assays

  • Liposome binding assays with fluorescently-labeled protein

  • Monolayer insertion experiments

• Functional screening:

  • Test for common biochemical activities (ATPase, GTPase, protease)

  • Assess ion channel properties in reconstituted systems

  • Evaluate peptide transport capabilities

Stage 3: Cellular and Molecular Biology Approaches

• Gene deletion/complementation:

  • Generate knockout mutants in Haemophilus influenzae

  • Phenotypic characterization under various conditions

  • Complementation studies with wild-type or mutant variants

• Localization studies:

  • Immunofluorescence microscopy

  • Subcellular fractionation

  • Protease accessibility assays to determine membrane topology

• Expression regulation analysis:

  • qRT-PCR under different conditions

  • Reporter gene assays

  • ChIP-seq to identify transcriptional regulators

These methods should be applied iteratively, with each experiment informing the design of subsequent investigations. For membrane proteins like HI_1736, particular attention should be paid to maintaining native membrane environment during functional assays.

How might HI_1736 contribute to Haemophilus influenzae pathogenesis based on its predicted properties?

While the specific role of HI_1736 in pathogenesis remains undetermined, its predicted membrane localization and sequence features suggest several potential contributions to Haemophilus influenzae virulence:

• Membrane Integrity and Stress Adaptation: Small membrane proteins often contribute to membrane stability under varying environmental conditions. HI_1736 may help maintain membrane integrity during infection, particularly when exposed to host-derived antimicrobial compounds or pH fluctuations in different anatomical niches.

• Host-Pathogen Interactions: The predicted transmembrane topology suggests HI_1736 could potentially:

  • Participate in adhesion to host cells or extracellular matrix components

  • Form part of secretion systems delivering virulence factors

  • Function in sensing host environmental cues and transducing signals

• Immune Evasion Mechanisms: Membrane proteins can contribute to evading host immune responses through:

  • Antigenic variation (though the high conservation might argue against this)

  • Masking surface structures recognized by immune receptors

  • Interfering with complement deposition or phagocytosis

• Relevance to Clinical Presentations: Recent case reports of Haemophilus influenzae serotype a (Hia) causing intracranial infections in pediatric patients highlight the continued clinical significance of this pathogen . While those reports don't specifically mention HI_1736, they emphasize the importance of understanding all components that might contribute to invasive disease potential.

Experimental approaches to evaluate these hypotheses include:

• Comparative expression analysis between invasive and non-invasive isolates

• Investigation of HI_1736 expression in animal infection models

• Evaluation of the impact of HI_1736 deletion on biofilm formation and antibiotic resistance

• Assessment of HI_1736 antigenic properties and exposure to the host immune system

A table summarizing potential pathogenic roles with corresponding experimental approaches would be valuable for research planning:

What is the evolutionary significance of HI_1736 across different Haemophilus species and related bacteria?

Understanding the evolutionary context of HI_1736 provides valuable insights into its biological importance and potential function:

Methodological approaches to investigate these evolutionary aspects include comparative genomics across diverse Haemophilus isolates, ancestral sequence reconstruction, and molecular clock analyses to estimate the age of this gene family. For HI_1736, which appears to be a small membrane protein with hydrophobic characteristics , evolutionary patterns may reveal whether it represents an ancient conserved function or a more recently acquired adaptive trait.

What are the optimal storage and handling conditions for maintaining recombinant HI_1736 stability?

Proper storage and handling of recombinant HI_1736 protein is critical for maintaining structural integrity and functional activity. Based on manufacturer recommendations and protein characteristics, the following protocol optimizes stability:

Long-term Storage Guidelines:

• Physical Form: Store as lyophilized powder whenever possible for maximum stability .

• Temperature: Maintain at -20°C/-80°C for long-term storage .

• Aliquoting Strategy: Upon receipt, prepare small single-use aliquots before freezing to avoid repeated freeze-thaw cycles .

Reconstitution Protocol:

• Pre-Handling: Briefly centrifuge the vial prior to opening to collect material at the bottom .

• Reconstitution Buffer: Use deionized sterile water to reconstitute to a concentration of 0.1-1.0 mg/mL .

• Protective Additives: For reconstituted protein, add glycerol to a final concentration of 5-50% (with 50% being the default recommendation) to prevent freeze-thaw damage .

• Buffer Composition: The protein is provided in Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which should be maintained in working solutions .

Working Stock Management:

• Short-term Storage: Working aliquots can be stored at 4°C for up to one week .

• Freeze-Thaw Limitations: Repeated freezing and thawing is strongly discouraged as it leads to protein denaturation .

• Temperature Transitions: When transitioning from frozen storage, thaw gradually on ice to minimize structure disruption.

Stability Monitoring:

• Visual Inspection: Check for precipitation or cloudiness before use.

• Activity Assessment: Periodically verify protein integrity using SDS-PAGE .

• Documentation: Maintain records of storage conditions and freeze-thaw cycles for each lot.

These guidelines are particularly important for membrane-associated proteins like HI_1736, which typically have greater stability challenges than soluble proteins due to their hydrophobic surfaces and tendency to aggregate when removed from their native membrane environment.

What experimental controls should be included when investigating potential functions of HI_1736?

Rigorous experimental design for investigating HI_1736 function requires appropriate controls to ensure valid and reproducible results:

Protein Quality Controls:

• Purity Verification: SDS-PAGE analysis confirming >90% purity .

• Identity Confirmation: Western blot with anti-His antibodies or mass spectrometry verification.

• Negative Control Protein: A similarly sized, His-tagged control protein with no expected activity in the system being tested.

• Denatured Protein Control: Heat-denatured HI_1736 to distinguish specific from non-specific effects.

Genetic/Molecular Controls:

• Vector Control: Empty expression vector for distinguishing protein-specific from vector-derived effects.

• Knockout/Complementation Pair: HI_1736 deletion mutant paired with complemented strain carrying the wild-type gene.

• Site-Directed Mutants: Proteins with mutations in predicted functional residues to establish structure-function relationships.

• Tagged Variant Controls: Compare His-tagged and untagged versions to assess tag interference with function.

Functional Assay Controls:

• Positive Control Systems: Well-characterized proteins with similar predicted functions.

• Dose-Response Assessment: Multiple protein concentrations to establish specific activity relationships.

• Time Course Analysis: Temporal measurements to distinguish primary from secondary effects.

• Buffer/Additive Controls: Test of buffer components without protein to rule out non-specific effects.

Cell-Based Experiment Controls:

• Wild-Type Comparison: Parental Haemophilus influenzae strain as baseline.

• Heterologous Expression Controls: Expression in different bacterial hosts to assess conservation of function.

• Conditional Expression Systems: Inducible promoters to control timing and level of expression.

• Cellular Localization Controls: Fractionation quality controls and markers for different cellular compartments.

Statistical and Experimental Design Controls:

• Biological Replicates: Independent experiments with different protein preparations.

• Technical Replicates: Multiple measurements within each experiment.

• Blinding Procedures: Where applicable, blind analysis of results to prevent bias.

• Randomization: Random assignment of samples to experimental conditions.

These comprehensive controls ensure that any functional properties attributed to HI_1736 are specific, reproducible, and biologically relevant rather than artifacts of experimental conditions or contamination.

What specialized techniques are required for membrane protein reconstitution of HI_1736?

Reconstitution of membrane proteins like HI_1736 into artificial membrane systems requires specialized techniques to maintain native structure and function:

Preparation of Purified HI_1736:

• Detergent Selection: Critical for initial solubilization - mild detergents such as DDM (n-Dodecyl β-D-maltoside), LMNG (Lauryl Maltose Neopentyl Glycol), or CHAPS are preferred to preserve protein structure.

• Protein:Detergent Ratio Optimization: Determine the minimal detergent concentration that maintains protein solubility without excessive detergent micelles.

• Buffer Composition: Maintain pH 8.0 as specified in the product information , with appropriate ionic strength (typically 150mM NaCl) and stabilizing agents such as glycerol.

Lipid System Selection:

• Lipid Composition: Consider using E. coli polar lipid extract or synthetic mixtures mimicking bacterial membranes (POPE/POPG mixtures).

• Lipid:Protein Ratio Determination: Typically starting with molar ratios of 200:1 to 1000:1 lipid:protein, with optimization required.

• Preparation of Liposomes: Small unilamellar vesicles (SUVs) prepared by sonication or extrusion through polycarbonate membranes (100nm pore size).

Reconstitution Methods Comparison:

Functional Verification Techniques:

• Proteoliposome Characterization:

  • Dynamic light scattering to confirm size distribution

  • Freeze-fracture electron microscopy to visualize protein incorporation

  • Sucrose density gradient centrifugation to separate empty liposomes

• Orientation Assessment:

  • Protease protection assays to determine topology

  • Antibody accessibility in intact vs. permeabilized vesicles

  • Chemical labeling of accessible residues

• Functional Assays:

  • Ion flux measurements if channel activity is suspected

  • Binding assays with fluorescently labeled ligands

  • Structural integrity verification by circular dichroism

These specialized techniques require careful optimization for HI_1736 specifically, as each membrane protein has unique requirements for successful reconstitution. The hydrophobic nature of HI_1736 indicated by its amino acid sequence suggests it will require particular attention to detergent selection and removal strategies to maintain native folding.