Recombinant Desulfitobacterium hafniense UPF0316 protein DSY1893 (DSY1893)

What is Desulfitobacterium hafniense DSY1893 protein?

Desulfitobacterium hafniense UPF0316 protein DSY1893 is a 174-amino acid protein (Q24WB0) that has been characterized in Desulfitobacterium hafniense. The recombinant version is typically expressed with an N-terminal histidine tag, allowing for efficient purification through immobilized metal affinity chromatography. The protein belongs to the UPF0316 family, whose members are characterized by conserved sequences but have not yet been fully functionally annotated . The amino acid sequence of DSY1893 is: MGSILQFVLIIITINITYVTLTTIRFILMIKGMRVYASLLSVLEVFIYIMGLSIILDNLDSYWNIAAYCCGYGVGVYLGSRIEERLALGYIMAQVIVECEYQGLAGELRDAGFGVTSWLGEGKTGPRMVMMVLAKRNRQKELLNRIDSLCSNAFVIFEEPKNFRGGFWAKKVLH .

What expression systems are effective for recombinant production of DSY1893?

DSY1893 protein can be successfully expressed in Escherichia coli expression systems with an N-terminal histidine tag . When working with recombinant D. hafniense proteins, heterologous expression has proven effective, as demonstrated with other D. hafniense proteins such as reductive dehalogenases. For instance, the tetrachloroethene (PCE) reductive dehalogenase (PceA) from D. hafniense strain Y51 has been successfully expressed in Shimwellia blattae, a Gram-negative gammaproteobacterium . For optimal expression of D. hafniense proteins, researchers should consider:

• Co-expression with appropriate chaperones (e.g., PceT for PceA enzymes)

• Addition of cofactor precursors such as 5,6-dimethylbenzimidazole and hydroxocobalamin

• Selection of appropriate antibiotic markers for plasmid retention

• Optimization of induction conditions specific to the expression system

What are the optimal storage conditions for maintaining DSY1893 stability?

Recombinant DSY1893 protein is typically supplied as a lyophilized powder. For storage, it is recommended to avoid repeated freezing and thawing cycles. Working aliquots should be stored at 4°C for up to one week to maintain optimal protein integrity and activity . For long-term storage, maintaining the protein in its lyophilized state or preparing appropriate buffer conditions with stabilizing agents would be advisable, similar to protocols used for other recombinant proteins.

How can researchers assess the potential role of DSY1893 in electron transport processes?

To investigate the potential role of DSY1893 in electron transport, researchers can employ methodologies similar to those used for studying complex I-like enzymes in D. hafniense. These approaches include:

• Inhibitor studies using specific electron transport inhibitors such as rotenone or piericidin A

• Growth experiments under different electron donor/acceptor combinations to observe metabolic impacts

• Comparative proteomic analysis to measure relative abundance under varying conditions

• Gene knockout or silencing experiments followed by phenotypic characterization

For example, research on the complex I-like enzyme in D. hafniense DCB-2 utilized rotenone inhibition studies under various growth conditions (pyruvate/fumarate, lactate/fumarate, pyruvate-only, lactate/ClOHPA, hydrogen/fumarate, and hydrogen/ClOHPA). Growth was monitored by measuring cell density at 600 nm over time . Similar approaches could be applied to understand DSY1893's potential involvement in electron transport processes.

What techniques are most effective for studying protein-protein interactions involving DSY1893?

To investigate protein-protein interactions involving DSY1893, researchers can employ several complementary techniques:

• Pull-down assays using the His-tagged recombinant protein as bait

• Co-immunoprecipitation studies with antibodies specific to DSY1893

• Bacterial two-hybrid systems for in vivo interaction studies

• Cross-linking mass spectrometry to identify interaction partners

• Surface plasmon resonance for quantifying binding kinetics

These approaches could help identify potential interactions between DSY1893 and other proteins in D. hafniense, such as those involved in redox processes or membrane functions, similar to studies that identified potential redox partners for the complex I-like enzyme .

How might DSY1893 function differ under varying electron donor and acceptor conditions?

These findings suggest that D. hafniense proteins may have differential expression or function depending on the energy metabolism employed. To investigate DSY1893's potential role under varying electron donor/acceptor conditions, researchers should:

• Design comparative growth experiments with different electron donors (pyruvate, lactate, hydrogen) and acceptors (fumarate, chlorophenols, PCE)

• Perform proteomic analysis to quantify DSY1893 abundance under each condition

• Conduct activity assays with purified DSY1893 in the presence of different potential substrates

• Compare transcriptomic data across growth conditions to identify co-regulated genes

What experimental approaches can address the membrane association potential of DSY1893?

Based on the amino acid sequence of DSY1893, which contains hydrophobic regions potentially indicative of membrane association, researchers might want to investigate its subcellular localization:

• Membrane fractionation studies followed by Western blot analysis

• Fluorescent protein tagging and microscopy for localization studies

• Membrane protein extraction methods optimized for D. hafniense

• Bioinformatic analysis of the amino acid sequence for transmembrane domains

• Lipid binding assays with purified DSY1893

The sequence "MGSILQFVLIIITINITYVTLTTIRFILMIKGMRVYASLLSVLEVFIYIMGLSIILDNLD" from the N-terminal portion of DSY1893 contains stretches of hydrophobic residues that may suggest membrane interaction potential .

How can researchers overcome expression challenges for DSY1893 in heterologous systems?

When expressing D. hafniense proteins heterologously, several challenges may arise. Based on experiences with other D. hafniense proteins such as reductive dehalogenases, the following strategies may improve DSY1893 expression:

• Co-expression with molecular chaperones to enhance proper folding

• Optimization of induction parameters (temperature, inducer concentration, duration)

• Use of specialized E. coli strains designed for membrane or difficult-to-express proteins

• Addition of appropriate cofactors or precursors to the culture medium

• Codon optimization of the DSY1893 gene for the expression host

For example, the formation of catalytically active PceA enzyme from D. hafniense Y51 in S. blattae was significantly enhanced when its dedicated PceT chaperone was co-produced and when 5,6-dimethylbenzimidazole and hydroxocobalamin were added to the cultures . Similar approaches might benefit DSY1893 expression.

What controls should be included in functional assays involving DSY1893?

When designing functional assays for DSY1893, researchers should include:

• Negative controls using denatured DSY1893 protein

• Positive controls with known enzymatic activities if functional predictions exist

• Buffer-only controls to account for non-specific reactions

• Controls with structurally similar proteins from related organisms

• Time-course experiments to establish reaction kinetics

• Temperature and pH optimization controls

These controls will help researchers distinguish between specific DSY1893 activities and background reactions, ensuring reliable and reproducible results.

How should researchers design experiments to identify the physiological role of DSY1893?

To identify the physiological role of DSY1893, a multi-faceted experimental approach is recommended:

• Gene knockout or knockdown studies in D. hafniense

• Phenotypic characterization of mutants under various growth conditions

• Complementation studies to confirm phenotype-genotype relationships

• Proteomic analysis comparing wild-type and mutant strains

• Metabolomic profiling to identify altered metabolic pathways

• Growth experiments under varying stress conditions

This approach follows the methodology used to investigate the role of complex I-like enzyme in D. hafniense DCB-2, where growth was monitored under different conditions in the presence and absence of specific inhibitors .

What analytical techniques are most appropriate for studying DSY1893 structure-function relationships?

These analytical techniques can provide insights into the structural features of DSY1893 that might determine its function, similar to the structural analyses performed for other D. hafniense proteins.

How does DSY1893 research fit into broader studies of D. hafniense metabolism?

D. hafniense demonstrates remarkable metabolic versatility, capable of utilizing various electron donors (pyruvate, lactate, hydrogen) and acceptors (fumarate, organohalogens). Research on DSY1893 fits into the broader context of understanding:

• The unique respiratory systems of D. hafniense, including its complex I-like enzyme

• Mechanisms of energy conservation under different electron donor/acceptor combinations

• The molecular basis for D. hafniense's ability to perform organohalide respiration

• Adaptations for survival in environments contaminated with halogenated compounds

For instance, studies have shown that D. hafniense strain DCB-2 relies on its complex I-like enzyme for growth with organic electron donors but can bypass this dependence when using hydrogen as an electron donor . Understanding DSY1893's role in this metabolic network could provide valuable insights into D. hafniense's physiological adaptations.

What emerging technologies are most promising for advancing DSY1893 research?

Several emerging technologies show promise for advancing DSY1893 research:

• CRISPR-Cas9 genome editing for precise genetic manipulation of D. hafniense

• Single-cell proteomics to analyze protein abundance in individual cells

• Cryo-electron microscopy for high-resolution structural studies

• Microfluidic techniques for real-time monitoring of cellular responses

• Advanced bioinformatics approaches for predicting protein function based on sequence and structural data

• Systems biology approaches to integrate multi-omics data for comprehensive metabolic modeling

These technologies could overcome current limitations in studying proteins like DSY1893 whose functions remain to be fully elucidated.