Recombinant Haemophilus influenzae UPF0761 membrane protein CGSHiEE_01670 (CGSHiEE_01670)

What is the UPF0761 membrane protein CGSHiEE_01670 from Haemophilus influenzae?

The UPF0761 membrane protein CGSHiEE_01670 is a 269 amino acid transmembrane protein from Haemophilus influenzae, a Gram-negative, facultatively anaerobic pathogenic bacterium belonging to the Pasteurellaceae family. This protein belongs to the uncharacterized protein family UPF0761 and is likely involved in membrane-associated functions. Haemophilus influenzae is responsible for a range of localized and invasive infections, and was the first free-living organism to have its entire genome sequenced . The protein has been recombinantly produced with an N-terminal His-tag for research purposes .

What are the biochemical properties of CGSHiEE_01670 that researchers should consider?

Researchers should consider several key biochemical properties when working with this membrane protein:

• Hydrophobicity profile: The protein contains multiple hydrophobic regions typical of transmembrane segments, which influences solubility and handling requirements.

• Membrane localization: As a membrane protein, it requires special considerations for maintaining native conformation during extraction and analysis.

• Secondary structure: Based on sequence analysis, the protein likely has multiple transmembrane alpha-helical domains.

• Stability considerations: The protein may require specific buffer compositions containing detergents or lipids to maintain stability once extracted from the membrane.

• Post-translational modifications: While expressed in E. coli, researchers should be aware that native post-translational modifications might be absent in the recombinant version.

The hydrophobic nature of this protein means standard protein handling protocols may need modification to prevent aggregation and maintain proper folding .

What are the optimal conditions for expressing recombinant CGSHiEE_01670?

Optimal expression of recombinant CGSHiEE_01670 has been achieved using the following protocol:

• Expression system: E. coli has been successfully used for heterologous expression .

• Expression construct: The protein is typically expressed with an N-terminal His-tag to facilitate purification.

• Culture conditions: Standard E. coli culture conditions with IPTG induction are suitable, but researchers should optimize temperature (often lowered to 16-18°C during induction) to enhance proper folding of membrane proteins.

• Induction parameters: For membrane proteins, slower expression at lower temperatures (16-18°C) for longer periods (overnight) often yields better results than rapid expression at 37°C.

• Media supplementation: Addition of glycerol (0.5-1%) can help stabilize membrane proteins during expression.

As with many membrane proteins, expression levels may be lower than soluble proteins, and optimization of conditions is crucial for maximizing yield while maintaining proper folding .

What purification strategy is most effective for isolating CGSHiEE_01670?

The most effective purification strategy for CGSHiEE_01670 involves:

• Cell lysis: Gentle lysis methods using specialized detergents rather than sonication alone are preferable for membrane proteins.

• Membrane fraction isolation: Differential centrifugation to isolate membrane fractions, followed by solubilization with appropriate detergents.

• Affinity chromatography: His-tag affinity purification using Ni-NTA resin is the primary purification step .

• Detergent considerations: Critical micelle concentration (CMC) must be maintained throughout the purification process to keep the protein soluble.

• Buffer components: Inclusion of 6% trehalose in Tris/PBS-based buffer at pH 8.0 has been used successfully for storage .

• Storage recommendations: The purified protein is typically stored as a lyophilized powder and can be reconstituted to 0.1-1.0 mg/mL in deionized sterile water with 5-50% glycerol for long-term storage at -20°C/-80°C .

For working aliquots, storage at 4°C for up to one week is recommended, with repeated freeze-thaw cycles being discouraged to maintain protein integrity .

How can researchers verify the purity and integrity of isolated CGSHiEE_01670?

Researchers should employ multiple complementary techniques to verify protein purity and integrity:

• SDS-PAGE analysis: The standard method for assessing purity, with expected purity greater than 90% as determined by SDS-PAGE .

• Western blotting: Using anti-His antibodies to confirm identity of the protein.

• Mass spectrometry: For accurate molecular weight determination and verification of sequence integrity.

• Circular dichroism (CD) spectroscopy: To assess secondary structure content and proper folding.

• Size-exclusion chromatography: To evaluate oligomeric state and detect potential aggregation.

• Dynamic light scattering: To assess homogeneity of the protein preparation.

For membrane proteins specifically, native gel electrophoresis in the presence of mild detergents can provide information about the oligomeric state in a membrane-mimicking environment.

What structural information is currently available for UPF0761 membrane proteins?

The UPF0761 family of membrane proteins, including CGSHiEE_01670, has been part of large-scale structure determination efforts. According to search results:

• Co-evolution analysis: These proteins have been analyzed using Rosetta co-evolution-guided structure prediction protocols, which leverage evolutionary sequence co-variation to predict protein structure .

• Predicted topology: The amino acid sequence suggests multiple transmembrane segments typical of integral membrane proteins.

• Structural homology: While specific structural data for CGSHiEE_01670 is limited, the UPF0761 family has been included in studies comparing accuracy of structural prediction methods, with some models achieving TMscores of 0.9, indicating high confidence predictions .

What are effective methods for determining the membrane topology of CGSHiEE_01670?

Researchers can employ several complementary approaches to determine membrane topology:

• Computational prediction: Using algorithms specifically designed for transmembrane proteins such as TMHMM, Phobius, or TOPCONS to predict topology based on hydrophobicity analysis.

• Cysteine scanning mutagenesis: Systematically replacing residues with cysteine and using membrane-impermeable thiol-reactive reagents to identify exposed regions.

• Protease protection assays: Limited proteolysis of membrane vesicles to identify protease-accessible regions.

• Reporter fusion constructs: Creating fusion proteins with reporters like GFP or alkaline phosphatase at various positions to determine orientation relative to the membrane.

• Surface labeling techniques: Using membrane-impermeable biotinylation reagents to identify surface-exposed domains.

For membrane proteins like CGSHiEE_01670, combining computational predictions with at least two experimental approaches is recommended for reliable topology determination.

How can researchers approach structural studies of CGSHiEE_01670 using advanced techniques?

Advanced structural studies of CGSHiEE_01670 can be approached through:

• X-ray crystallography:

  • Requires screening numerous detergents or lipidic cubic phase conditions

  • Often necessitates construct optimization to remove flexible regions

  • May benefit from fusion partners that facilitate crystallization

• Cryo-electron microscopy:

  • Particularly useful for larger membrane protein complexes

  • Sample preparation in nanodiscs or amphipols can preserve native-like environment

  • Can reveal structural details without crystallization

• NMR spectroscopy:

  • Solution NMR requires isotopic labeling and detergent optimization

  • Solid-state NMR can be performed on protein reconstituted in lipid bilayers

  • Provides dynamic information not accessible by static methods

• Co-evolutionary analysis and molecular modeling:

  • Leverages multiple sequence alignments to identify co-evolving residues

  • Integration with Rosetta has shown promising results for membrane proteins

  • Models can guide experimental design and interpretation

• Hydrogen-deuterium exchange mass spectrometry:

  • Provides information about solvent-accessible regions

  • Can track conformational changes in different conditions

Success in structural studies often requires screening multiple constructs with various tags, expression conditions, and membrane-mimetic environments .

What is known about the function of UPF0761 membrane proteins in Haemophilus influenzae?

• Membrane localization: As integral membrane proteins, they likely participate in membrane-associated functions such as transport, signaling, or maintaining membrane integrity.

• Conservation: The protein is conserved across different H. influenzae strains, suggesting functional importance .

• Related membrane proteins: Research on other H. influenzae membrane proteins has shown that some function as porins, facilitating the entry of hydrophilic molecules . The 40 kDa major outer-membrane protein in H. influenzae has been demonstrated to function as a porin, with mutant strains lacking this protein showing decreased permeability to antibiotics and sugars .

• Sequence analysis: While not directly stated for CGSHiEE_01670, sequence analysis of the UPF0761 family suggests potential roles in transport or membrane structural organization.

How can researchers design experiments to determine the functional role of CGSHiEE_01670?

To elucidate the functional role of CGSHiEE_01670, researchers can implement a systematic approach:

• Gene knockout studies:

  • Create deletion mutants in H. influenzae

  • Assess growth rates, morphology, and response to various stressors

  • Compare permeability to various compounds between wild-type and mutant strains, similar to studies performed with other H. influenzae membrane proteins

• Protein-protein interaction studies:

  • Perform pull-down assays using His-tagged CGSHiEE_01670

  • Employ crosslinking methods followed by mass spectrometry

  • Use bacterial two-hybrid systems to identify interaction partners

• Transport studies:

  • Reconstitute purified protein in liposomes

  • Assess transport of various substrates using fluorescent indicators or radioisotopes

  • Compare substrate specificity and kinetics with known transporters

• Localization studies:

  • Use immunofluorescence microscopy to determine precise subcellular localization

  • Investigate co-localization with other membrane proteins of known function

• Expression analysis:

  • Determine expression levels under various growth conditions

  • Identify transcriptional regulators controlling gene expression

  • Analyze co-expression patterns with functionally related genes

A combination of these approaches would provide complementary insights into the functional role of CGSHiEE_01670 .

How might CGSHiEE_01670 contribute to pathogenicity or antibiotic resistance in H. influenzae?

While specific information about CGSHiEE_01670's role in pathogenicity is not directly provided in the search results, we can draw insights from related research on H. influenzae membrane proteins:

• Potential role in antibiotic permeability: Similar to the 40 kDa porin in H. influenzae, CGSHiEE_01670 might influence bacterial permeability to antibiotics. Studies have shown that strains with decreased quantities of certain outer membrane proteins had reduced permeability to chloramphenicol and other hydrophilic antibiotics .

• Host interaction: Membrane proteins often mediate interactions with host cells. As observed in research on Staphylococcus proteins, bacterial surface proteins can show differential abundance following host cell colonization .

• Immune evasion: Membrane proteins may participate in mechanisms that help bacteria evade host immune responses.

• Biofilm formation: Many membrane proteins contribute to bacterial adhesion and biofilm formation, which enhance antibiotic resistance and persistence.

• Environmental adaptation: Membrane proteins help bacteria adapt to changing environmental conditions encountered during infection.

To investigate these possibilities, researchers could compare wild-type H. influenzae with strains overexpressing or lacking CGSHiEE_01670, challenging them with various antibiotics and host defense mechanisms to observe differential responses .

How does CGSHiEE_01670 compare with homologous proteins in other bacterial species?

Comparative analysis of CGSHiEE_01670 with homologous proteins reveals:

• Conservation patterns: The UPF0761 family appears in multiple bacterial species, with varying degrees of sequence conservation. This conservation suggests functional importance despite the "uncharacterized" designation.

• Evolutionary relationships: Phylogenetic analysis can reveal how these proteins have evolved across bacterial species and identify key conserved residues likely essential for function.

• Structural similarities: Homology modeling based on known structures of related proteins can inform structural predictions for CGSHiEE_01670.

• Functional insights: Functional annotations of homologs in better-characterized species may provide clues to CGSHiEE_01670's function.

The search results indicate that large-scale studies have included the YIHY: UPF0761 membrane protein (which appears to be related to CGSHiEE_01670) in comparative analyses, with multiple sequence alignments generating thousands of sequences (10,144 sequences mentioned in search result ).

What bioinformatic approaches are most suitable for analyzing the CGSHiEE_01670 protein family?

The most appropriate bioinformatic approaches for analyzing the CGSHiEE_01670 protein family include:

• Multiple sequence alignment: Tools like MUSCLE, MAFFT, or Clustal Omega can identify conserved regions across homologs.

• Co-evolutionary analysis: Methods leveraging evolutionary sequence co-variation have proven valuable for structural prediction of UPF0761 family proteins :

  • The Rosetta co-evolution-guided structure prediction protocol has been applied to this family

  • Evolutionary coupling analysis can identify residues that co-evolve, suggesting spatial proximity in the folded structure

• Transmembrane topology prediction: Specialized algorithms for membrane proteins such as TMHMM, TOPCONS, or Phobius can predict membrane-spanning regions.

• Functional domain identification: Tools like InterProScan can identify conserved domains that might suggest function.

• Phylogenetic analysis: Building phylogenetic trees to understand evolutionary relationships within the protein family.

Research has demonstrated that co-evolutionary analysis combined with modeling can achieve high confidence predictions for membrane proteins like those in the UPF0761 family, with TMscore values of 0.9 reported in large-scale studies .

How can researchers use CGSHiEE_01670 as a model system for studying membrane protein biogenesis?

CGSHiEE_01670 presents an opportunity to study membrane protein biogenesis in gram-negative bacteria:

• Targeting and insertion mechanisms: Researchers can investigate how CGSHiEE_01670 is targeted to and inserted into the membrane by:

  • Creating fusion constructs with fluorescent tags

  • Performing in vitro translation/translocation assays

  • Studying interactions with membrane insertion machinery components

• Folding kinetics: The folding pathway of CGSHiEE_01670 can be studied using:

  • Single-molecule techniques to track folding events

  • Hydrogen-deuterium exchange to identify folding intermediates

  • Cysteine accessibility methods to monitor conformational changes

• Lipid interactions: The role of specific lipids in proper folding can be investigated by:

  • Reconstituting the protein in defined lipid compositions

  • Performing lipid binding assays

  • Analyzing how lipid environment affects stability and function

• Quality control mechanisms: Researchers can explore how misfolded variants are recognized and degraded by:

  • Creating destabilizing mutations

  • Tracking protein degradation pathways

  • Identifying quality control components that interact with the protein

These approaches would contribute to the broader understanding of membrane protein biogenesis beyond this specific protein.

What methods can be used to investigate the role of CGSHiEE_01670 in bacterial membrane permeability?

To investigate the potential role of CGSHiEE_01670 in membrane permeability, researchers can employ methods similar to those used for other H. influenzae membrane proteins :

• Liposome swelling assays:

  • Reconstitute purified protein in liposomes

  • Measure changes in liposome size when exposed to different solutes

  • Calculate permeability coefficients for various molecules

• Antibiotic susceptibility testing:

  • Compare minimum inhibitory concentrations between wild-type and CGSHiEE_01670-deficient strains

  • Assess the rate of antibiotic accumulation using fluorescent or radiolabeled antibiotics

  • Study the effect of overexpression on antibiotic resistance profiles

• Electrophysiological measurements:

  • Perform patch-clamp analysis on proteoliposomes containing CGSHiEE_01670

  • Measure ion conductance and selectivity

  • Characterize channel gating properties if applicable

• Fluorescent probe studies:

  • Use membrane-impermeable fluorescent dyes to assess changes in membrane permeability

  • Track dye uptake in real-time using fluorescence microscopy or spectroscopy

• Sugar permeability assays:

  • Similar to studies with other H. influenzae membrane proteins, measure permeability to sucrose and raffinose

  • Compare growth rates on different carbon sources between wild-type and mutant strains

Research on other H. influenzae membrane proteins has demonstrated that these approaches can effectively characterize their roles in permeability, revealing functional relationships to growth rate and antibiotic resistance .

How can CGSHiEE_01670 be used in developing new antimicrobial strategies against H. influenzae?

CGSHiEE_01670 could be leveraged for antimicrobial development through several research approaches:

• Target-based drug discovery:

  • If CGSHiEE_01670 proves essential for bacterial viability, it could serve as a direct drug target

  • High-throughput screening against the purified protein could identify inhibitory compounds

  • Structure-based drug design could optimize lead compounds for potency and specificity

• Permeability enhancement:

  • If the protein functions in membrane permeability, compounds that alter its function could enhance uptake of existing antibiotics

  • Combination therapy approaches could be developed to overcome resistance mechanisms

• Diagnostic applications:

  • Antibodies against surface-exposed epitopes could be developed for rapid identification of H. influenzae

  • Point-of-care testing could improve treatment selection and antimicrobial stewardship

• Vaccine development considerations:

  • If surface-exposed, conserved epitopes are identified, CGSHiEE_01670 could be evaluated as a vaccine antigen

  • Recombinant protein or peptide-based vaccines could be developed and tested in animal models

• Drug delivery systems:

  • Understanding the structure and function could inform development of targeted drug delivery systems that interact specifically with H. influenzae membranes

These approaches would require thorough characterization of CGSHiEE_01670's structure, function, and essentiality in H. influenzae before proceeding to translational applications.

What are the main challenges in working with recombinant membrane proteins like CGSHiEE_01670?

Researchers face several significant challenges when working with recombinant membrane proteins like CGSHiEE_01670:

• Expression limitations:

  • Membrane proteins often express poorly due to toxicity, hydrophobicity, and folding issues

  • They may require specialized expression systems and careful optimization

• Solubilization hurdles:

  • Extracting membrane proteins while maintaining native structure requires suitable detergents

  • Different detergents can significantly affect protein stability and activity

• Purification difficulties:

  • Membrane proteins tend to aggregate during purification

  • Detergent micelles can interfere with certain purification techniques

  • Mixed populations of oligomeric states are common

• Stability concerns:

  • Many membrane proteins are inherently unstable when removed from their native lipid environment

  • They may require specific lipids or lipid-like molecules to maintain function

• Analytical challenges:

  • Detergents can interfere with many analytical techniques

  • Size determination is complicated by detergent/lipid binding

  • Functional assays may be difficult to design and interpret

These challenges require specialized approaches and often result in lower yields compared to soluble proteins .

How can researchers overcome solubility and stability issues when working with CGSHiEE_01670?

To overcome solubility and stability challenges with CGSHiEE_01670, researchers can implement several strategies:

• Optimized buffer systems:

  • Include 6% trehalose in Tris/PBS-based buffer at pH 8.0 as used successfully for this protein

  • Add stabilizing agents such as glycerol (5-50%) for long-term storage

• Detergent screening:

  • Systematically test multiple detergent types (ionic, non-ionic, zwitterionic)

  • Evaluate detergent mixtures which can sometimes better mimic natural membrane environments

  • Consider mild detergents like digitonin or LMNG that preserve protein-protein interactions

• Membrane mimetics:

  • Nanodiscs composed of membrane scaffold proteins and phospholipids

  • Amphipols which can replace detergents for more stable preparations

  • Lipid cubic phases for crystallization and functional studies

• Fusion partners and engineering:

  • Addition of solubility-enhancing fusion partners beyond the His-tag

  • Targeted mutagenesis of aggregation-prone regions

  • Thermostabilizing mutations identified through scanning mutagenesis

• Storage considerations:

  • Lyophilization has been used successfully for CGSHiEE_01670

  • Avoid repeated freeze-thaw cycles

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

These approaches can be implemented iteratively, with protein quality assessed at each step using techniques like size-exclusion chromatography and activity assays.

What experimental controls are critical when studying the function of uncharacterized membrane proteins like CGSHiEE_01670?

When investigating uncharacterized membrane proteins like CGSHiEE_01670, several critical experimental controls must be included:

• Expression and purification controls:

  • Empty vector controls to account for host cell background

  • Different purification tags to ensure tag position doesn't affect function

  • Elution buffer-only controls to account for buffer components in functional assays

• Protein quality controls:

  • Size-exclusion chromatography to verify monodispersity

  • Circular dichroism to confirm proper folding

  • Thermal shift assays to assess stability under experimental conditions

• Functional assay controls:

  • Detergent-only controls to account for detergent effects on assay systems

  • Well-characterized membrane proteins as positive controls

  • Denatured protein as negative control

  • Dilution series to establish dose-dependency

• Specificity controls:

  • Structurally related but functionally distinct proteins

  • Site-directed mutants affecting key predicted functional residues

  • Competitive inhibition assays to confirm binding specificity

• System validation:

  • Complementation studies in knockout strains

  • Multiple independent methods to verify key findings

  • Careful statistical analysis with appropriate replicates

These controls help distinguish true protein functions from artifacts and provide confidence in results from uncharacterized protein studies.

How can researchers integrate structural, functional, and genomic data for comprehensive analysis of CGSHiEE_01670?

Comprehensive analysis of CGSHiEE_01670 requires integration of multiple data types:

• Data integration frameworks:

  • Establish pipelines that combine structural predictions, genomic context, and experimental data

  • Use machine learning approaches to identify patterns across heterogeneous datasets

  • Implement graph-based data structures to capture relationships between different data types

• Structural-functional correlation:

  • Map functional data onto structural models

  • Identify structure-function relationships through comparative analysis

  • Use evolutionary coupling data to predict functional sites

• Genomic context analysis:

  • Examine gene neighborhood to identify functionally related genes

  • Analyze co-expression patterns across different conditions

  • Investigate conservation patterns across related species

• Systems biology approaches:

  • Place CGSHiEE_01670 within protein-protein interaction networks

  • Model metabolic pathways potentially involving this protein

  • Integrate transcriptomic and proteomic data to understand regulation

• Visualization tools:

  • Develop custom visualization methods to represent integrated data

  • Use existing platforms like Cytoscape for network analysis

  • Create interactive visualizations that connect genomic position, structure, and function

The integration of co-evolutionary analysis with structural modeling has proven particularly powerful for membrane proteins like CGSHiEE_01670, as demonstrated by the high TMscores achieved in large-scale studies .

What computational resources and databases are most valuable for researchers studying CGSHiEE_01670?

Researchers studying CGSHiEE_01670 should utilize these computational resources and databases:

• Sequence databases:

  • UniProt: Contains annotated entries for CGSHiEE_01670 (A5UG94) and homologs (P44608)

  • InterPro: For domain identification and functional classification

  • Pfam: For protein family information on UPF0761

• Structural resources:

  • RCSB Protein Data Bank: Contains grouped information based on 95% sequence identity

  • AlphaFold DB: For structure predictions based on deep learning

  • SWISS-MODEL: For homology modeling of CGSHiEE_01670

• Membrane protein-specific tools:

  • TOPCONS: For consensus prediction of membrane protein topology

  • OPM database: For spatial orientations of proteins in membranes

  • MemProtMD: For molecular dynamics simulations in membranes

• Genomic context tools:

  • STRING: For protein-protein interaction networks

  • MicrobesOnline: For comparative genomics and gene neighborhood analysis

  • DOOR: For operon predictions in bacterial genomes

• Specialized prediction servers:

  • EVfold: For co-evolutionary analysis

  • PSIPRED: For secondary structure prediction

  • MemBrain: For transmembrane protein structure prediction

• Integrated analysis platforms:

  • Jalview: For sequence alignment visualization and analysis

  • PyMOL/Chimera: For structural visualization and analysis

  • Galaxy platform: For implementing reproducible analysis workflows

These resources collectively provide a comprehensive toolkit for investigating the structure, function, and evolution of CGSHiEE_01670 and related proteins.