Recombinant Acidovorax ebreus UPF0060 membrane protein Dtpsy_1668 (Dtpsy_1668)

What is Acidovorax ebreus and what is the taxonomic classification of this organism?

Acidovorax ebreus is a bacterial species first described by Byrne-Bailey et al. in 2010, though it has not been validly published according to the List of Prokaryotic Names with Standing in Nomenclature (LPSN) . The organism belongs to the genus Acidovorax, which was established by Willems et al. in 1990 .

The full taxonomic lineage of Acidovorax ebreus is:

• Domain: Bacteria

• Phylum: Proteobacteria

• Class: Betaproteobacteria

• Order: Burkholderiales

• Family: Comamonadaceae

• Genus: Acidovorax

• Species: Acidovorax ebreus

Acidovorax ebreus strain TPSY is notable as the first anaerobic nitrate-dependent Fe(II) oxidizer for which a complete genome sequence is available . The etymology of the genus name Acidovorax comes from Latin terms: "acidum" (acid) and "vorax" (voracious), referring to acid-devouring bacteria .

How is the recombinant form of Dtpsy_1668 protein typically produced for research purposes?

The recombinant form of Dtpsy_1668 is typically produced using standard protein expression systems. Based on commercial product information, the following methods are employed:

• Expression systems:

  • The protein is commonly expressed in bacterial systems, particularly E. coli

  • Alternative expression platforms include baculovirus expression systems

• Construct design:

  • The full-length protein (amino acids 1-110) is cloned into expression vectors

  • Often includes an N-terminal His-tag for purification purposes

  • Some versions may include different fusion tags determined during the manufacturing process

• Purification and preparation:

  • Purified to >85-90% purity as determined by SDS-PAGE

  • Typically prepared as a lyophilized powder

  • Storage buffer may contain Tris/PBS-based buffer with 6% Trehalose at pH 8.0

The recombinant protein is designed to facilitate research applications requiring pure protein preparations, such as structural studies, antibody production, or functional characterization.

What are the optimal storage and handling conditions for recombinant Dtpsy_1668 to maintain stability and functionality?

The optimal storage and handling conditions for recombinant Dtpsy_1668 are critical for maintaining protein stability and functionality in research applications. Based on manufacturer recommendations, the following guidelines should be followed:

Handling recommendations:

• Brief centrifugation of the vial prior to opening is recommended to bring contents to the bottom

• Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended by default) for long-term storage

• Aliquot to avoid repeated freeze-thaw cycles, which significantly reduce protein stability

The shelf life of the protein is influenced by multiple factors including storage state, buffer ingredients, storage temperature, and the intrinsic stability of the protein itself . Following these guidelines will help ensure the protein maintains its structure and functional properties for research applications.

What experimental approaches are appropriate for studying membrane localization and topology of Dtpsy_1668?

Studying the membrane localization and topology of membrane proteins like Dtpsy_1668 requires specialized experimental approaches due to their hydrophobic nature and integration into lipid bilayers. Based on standard methodologies for membrane protein analysis, the following approaches are recommended:

• Computational prediction methods:

  • Hydropathy analysis and transmembrane domain prediction using algorithms like TMHMM, Phobius, or TOPCONS

  • Sequence-based topology prediction tools can help identify membrane-spanning regions and orientation

• Biochemical approaches:

  • Protease protection assays to determine which regions are accessible from different sides of the membrane

  • Chemical labeling of accessible residues (e.g., cysteine scanning and labeling)

  • Glycosylation mapping using engineered glycosylation sites

• Structural biology methods:

  • Cryo-electron microscopy of membrane protein complexes

  • X-ray crystallography (challenging for membrane proteins)

  • NMR spectroscopy for smaller membrane proteins or domains

• Fluorescence-based approaches:

  • GFP-fusion proteins to track cellular localization

  • FRET (Förster Resonance Energy Transfer) to study proximity relationships

  • Fluorescence microscopy to visualize cellular distribution

• Immunological methods:

  • Immunogold electron microscopy to visualize the protein in native membranes

  • Immunofluorescence with domain-specific antibodies to determine topology

For Dtpsy_1668 specifically, its identification as a UPF0060 family member suggests it's likely a multi-pass membrane protein localized to the cell inner membrane , providing a starting point for experimental design.

What is known about the function and biological role of UPF0060 family proteins like Dtpsy_1668?

The UPF0060 protein family, to which Dtpsy_1668 belongs, remains relatively uncharacterized in terms of specific biological functions. The "UPF" designation (Uncharacterized Protein Family) itself indicates limited functional annotation. Based on available information:

• Structural characteristics:

  • UPF0060 family proteins are typically multi-pass membrane proteins

  • They are commonly localized to the cell inner membrane

  • The proteins are relatively small (Dtpsy_1668 is 110 amino acids)

• Genomic context:

  • In Acidovorax ebreus strain TPSY, Dtpsy_1668 is encoded within the genome of an organism described as "optimized for survival in a complex environmental system"

  • The strain is an anaerobic nitrate-dependent Fe(II) oxidizer, suggesting potential roles in environmental adaptation or metal metabolism

• Phylogenetic distribution:

  • Similar proteins exist across various bacterial species, particularly in proteobacteria

  • Conservation across species suggests a fundamental role in bacterial physiology

• Hypothesized functions:

  • Possible roles in membrane integrity or transport processes due to transmembrane localization

  • May contribute to adaptations for anaerobic respiration or metal oxidation processes given the organism's metabolism

  • Could function in stress response or environmental adaptation

Further experimental characterization is needed to determine the specific function of Dtpsy_1668 and related UPF0060 family proteins. Approaches might include gene knockout studies, protein-protein interaction analyses, or comparative genomics across diverse bacterial species harboring these proteins.

What experimental design would be appropriate to elucidate the potential role of Dtpsy_1668 in Fe(II) oxidation by Acidovorax ebreus?

Given that Acidovorax ebreus strain TPSY is an anaerobic nitrate-dependent Fe(II) oxidizer , investigating the potential role of Dtpsy_1668 in iron metabolism requires a comprehensive experimental approach:

Proposed Experimental Design:

• Gene expression analysis:

  • RT-qPCR to measure Dtpsy_1668 expression under varying iron conditions

  • RNA-Seq comparing transcriptomes in iron-rich vs. iron-limited conditions

  • Promoter analysis to identify potential iron-responsive regulatory elements

• Gene disruption studies:

  • CRISPR-Cas9 or homologous recombination to generate Dtpsy_1668 knockout strains

  • Complementation with wild-type and mutated versions to confirm phenotypes

  • Phenotypic characterization focusing on:

    • Growth rates under anaerobic Fe(II)-oxidizing conditions

    • Fe(II) oxidation rates using ferrozine assays

    • Nitrate reduction capacity

• Protein localization during Fe(II) oxidation:

  • Fluorescently tagged Dtpsy_1668 to track subcellular distribution

  • Membrane fractionation followed by Western blotting

  • Immunogold electron microscopy to visualize protein in relation to Fe(II) oxidation machinery

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify binding partners

  • Bacterial two-hybrid assays

  • Proximity labeling followed by mass spectrometry

• Metal binding assays:

  • Isothermal titration calorimetry (ITC) with purified protein

  • Metal-catalyzed oxidation (MCO) assays

  • Electron paramagnetic resonance (EPR) spectroscopy

• Structural changes upon Fe(II) binding:

  • Circular dichroism to detect secondary structure changes

  • Hydrogen-deuterium exchange mass spectrometry

  • X-ray absorption spectroscopy

This multi-faceted approach would provide complementary lines of evidence to determine whether Dtpsy_1668 plays a direct role in Fe(II) oxidation, functions as an accessory protein in the process, or has an unrelated function in membrane biology.

How might structural modeling and computational approaches be used to predict functional domains and potential interaction sites in Dtpsy_1668?

Structural modeling and computational approaches provide valuable insights for membrane proteins like Dtpsy_1668 where experimental structural determination can be challenging:

• Homology modeling workflow:

  • Template identification using PSI-BLAST against the PDB database

  • Sequence alignment optimization with membrane-specific substitution matrices

  • Model building using specialized membrane protein modeling tools (e.g., MEMOIR, MEDELLER)

  • Model refinement with molecular dynamics simulations in a membrane environment

  • Validation using tools like ProSA-web and PROCHECK

• Ab initio modeling approaches:

  • For novel folds with no suitable templates

  • Rosetta Membrane for de novo modeling in implicit membrane environments

  • AlphaFold2 or RoseTTAFold incorporation of co-evolutionary information

• Functional domain prediction:

  • Conserved domain analysis using CDD, PFAM, and InterPro

  • Evolutionary trace analysis to identify functionally important residues

  • Analysis of UPF0060 family alignments to identify conserved motifs

• Molecular dynamics simulations:

  • Explicit membrane simulations to study protein-lipid interactions

  • Assessment of stability in different membrane compositions

  • Water/ion permeation studies to identify potential channel function

• Ligand binding site prediction:

  • CASTp or COACH for pocket identification

  • Metal binding site prediction using MIB, TEMSP

  • Molecular docking with potential ligands, including iron-containing molecules

• Protein-protein interaction prediction:

  • ZDOCK or HADDOCK for docking with potential partners

  • Analysis of surface electrostatics and hydrophobicity

  • Coevolution-based contact prediction between proteins

The amino acid sequence of Dtpsy_1668 (MLPFKTLALFLLTAVAEIVGCYLPWLWLRQGRSAWLLVPAAASLALFAWLLTLHPAATGRVYAAYGGVYVAVALVWLWTVDGVRPGPWDWLGVSVTLCGMAIIAFAPRGG) contains hydrophobic stretches indicative of transmembrane regions, which would be emphasized in the structural modeling process. Integration of computational predictions with targeted experimental validation would provide testable hypotheses about Dtpsy_1668 function.

What research approaches would be suitable for investigating the phylogenetic distribution and evolutionary conservation of Dtpsy_1668 homologs across bacterial species?

Investigating the phylogenetic distribution and evolutionary history of Dtpsy_1668 homologs requires a systematic approach combining bioinformatics and comparative genomics:

Research Approach Framework:

• Homolog identification:

  • BLAST searches against comprehensive databases (NCBI nr, UniProt)

  • Profile-based searches using HMMer with UPF0060 family profiles

  • Positional ortholog identification using genome context conservation

  • Data collection from diverse bacterial phyla, emphasizing:

    • Close relatives in Acidovorax genus

    • Other members of Comamonadaceae family

    • Broader distribution in Proteobacteria

    • Potential distant homologs in other bacterial phyla

• Multiple sequence alignment construction:

  • Specialized membrane protein alignment tools (e.g., PRALINE-TM)

  • Manual refinement focusing on transmembrane region alignments

  • Incorporation of structural information where available

• Phylogenetic analysis:

  • Maximum likelihood methods (RAxML, IQ-TREE)

  • Bayesian inference approaches (MrBayes)

  • Selection of appropriate evolutionary models for membrane proteins

  • Bootstrap analysis and posterior probability assessment

• Evolutionary rate analysis:

  • Site-specific evolutionary rates using PAML

  • Identification of positively selected sites

  • Conservation analysis using ConSurf or Evolutionary Trace

• Genomic context analysis:

  • Examination of neighboring genes across species

  • Identification of conserved genomic neighborhoods

  • Detection of potential operonic structures

• Correlation with ecological and physiological traits:

  • Mapping presence/absence against metabolic capabilities

  • Correlation with habitat preferences (e.g., anaerobic environments)

  • Association with iron oxidation or other metal metabolism

This approach would elucidate the evolutionary history of Dtpsy_1668 and potentially provide functional insights based on patterns of conservation and co-evolution with other genes. The analysis might reveal whether homologs are specifically associated with anaerobic iron oxidation or have broader distribution suggesting alternative functions.

What are the critical factors to consider when designing experiments to study protein-lipid interactions for membrane proteins like Dtpsy_1668?

Studying protein-lipid interactions for membrane proteins like Dtpsy_1668 requires careful experimental design considering the protein's hydrophobic nature and its native lipid environment:

Critical Experimental Design Factors:

• Membrane mimetic selection:

  Mimetic System	Advantages	Limitations	Applicability to Dtpsy_1668
  Detergent micelles	Simple preparation, good for spectroscopy	May distort native structure	Initial solubilization and purification
  Bicelles	Disc-like, bilayer structure	Limited stability	NMR studies of structure
  Nanodiscs	Defined size, native-like bilayer	Complex assembly	Studying specific lipid interactions
  Liposomes	Enclosed bilayer, transport studies	Heterogeneous	Functional reconstitution assays
  Native nanodiscs	Preserves native lipids	Challenging isolation	Maintaining native interactions

• Lipid composition considerations:

  • Use of E. coli lipids for bacterial membrane proteins as a starting point

  • Systematic variation of lipid headgroups (PG, PE, CL) to identify preferences

  • Examination of acyl chain length and saturation effects

  • Testing of potential specific lipid interactions (e.g., cardiolipin)

• Protein purification strategies:

  • Gentle solubilization with mild detergents (DDM, LMNG)

  • Minimizing time in detergent micelles

  • Stabilization during purification (e.g., glycerol, specific lipids)

  • Careful tag placement to avoid interference with lipid interactions

• Biophysical techniques selection:

  • Fluorescence spectroscopy for monitoring lipid effects on structure

  • Differential scanning calorimetry for thermostability assessment

  • Microscale thermophoresis for binding studies

  • Native mass spectrometry for direct lipid binding detection

• Functional assessment approaches:

  • Lipid-dependent activity assays if function is known

  • Structural stability as a function of lipid environment

  • Oligomerization state monitoring in different lipid contexts

• Controls and validation:

  • Comparison with other membrane proteins of similar size

  • Use of lipid-binding mutants as negative controls

  • Multiple complementary techniques to confirm observations

For Dtpsy_1668 specifically, its 110-amino acid size and multi-pass membrane protein nature suggest nanodiscs or bicelles may be appropriate systems for detailed interaction studies, while initial characterization could employ detergent micelles for practical solubilization.

What research design would you propose to resolve contradictory results from different expression systems for recombinant Dtpsy_1668?

When faced with contradictory results from different expression systems for recombinant Dtpsy_1668, a systematic troubleshooting approach is necessary to identify the source of discrepancies and determine the most physiologically relevant findings:

Proposed Research Design:

• Systematic comparison of expression systems:

  • Direct comparison of protein expressed in E. coli , baculovirus , and other systems

  • Standardization of constructs (identical tags, cloning sites, and expression regions)

  • Comprehensive characterization of expression products:

    • SDS-PAGE and Western blot analysis

    • Mass spectrometry for exact mass determination

    • N-terminal sequencing to confirm processing

• Post-translational modification analysis:

  • Phosphorylation analysis by Pro-Q Diamond staining and LC-MS/MS

  • Glycosylation detection using glycoprotein staining methods

  • Comparison of modification patterns across expression systems

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to compare secondary structure

  • Intrinsic fluorescence to evaluate tertiary structure

  • Size-exclusion chromatography to determine oligomeric state

  • Limited proteolysis to assess domain folding

• Membrane incorporation studies:

  • Reconstitution into liposomes of defined composition

  • Proteoliposome flotation assays to confirm membrane integration

  • Freeze-fracture electron microscopy to visualize membrane incorporation

• Functional characterization:

  • Development of relevant functional assays based on predicted roles

  • Parallel testing of proteins from different sources

  • Quantitative comparison of activity metrics

• Native protein comparison:

  • If possible, isolation of native Dtpsy_1668 from Acidovorax ebreus

  • Direct comparison with recombinant versions

  • Complementation studies in knockout strains

Resolution Framework:

This comprehensive approach would identify the source of contradictions and establish which expression system produces the most native-like protein for further structural and functional studies of Dtpsy_1668.

What are the most promising future research directions for understanding the structure and function of Dtpsy_1668 in the context of Acidovorax ebreus biology?

Based on current knowledge, several promising research directions could significantly advance understanding of Dtpsy_1668 and its role in Acidovorax ebreus biology:

• Structural characterization:

  • Determination of high-resolution structure using cryo-EM or X-ray crystallography

  • Integration of computational predictions with experimental validation

  • Mapping of transmembrane topology and identification of functional domains

• Functional genomics approaches:

  • Generation of knockout and complementation strains

  • Transcriptomic analysis under varied environmental conditions (aerobic/anaerobic, iron availability)

  • Global protein interaction studies to place Dtpsy_1668 in cellular networks

• Connection to anaerobic iron oxidation:

  • Investigation of potential roles in the Fe(II) oxidation pathway of A. ebreus

  • Exploration of relationships with electron transport components

  • Studies of protein expression during active iron oxidation

• Comparative biology:

  • Analysis of UPF0060 family proteins across diverse bacterial species

  • Correlation of sequence variations with ecological niches and metabolic capabilities

  • Identification of conserved features that might indicate function

• Environmental adaptation mechanisms:

  • Examination of Dtpsy_1668 contribution to survival in complex environmental systems

  • Investigation of potential roles in stress response or adaptation

  • Studies of membrane dynamics in response to environmental changes