Recombinant Desulfitobacterium hafniense UPF0060 membrane protein DSY4157 (DSY4157)

What is DSY4157 and what organism does it originate from?

DSY4157 is a membrane protein that belongs to the UPF0060 protein family and originates from Desulfitobacterium hafniense. The protein is 108 amino acids in length and can be recombinantly expressed with a His-tag in E. coli expression systems . D. hafniense is a Gram-positive anaerobic bacterium with a versatile metabolism, capable of dehalogenation, metal reduction, and other diverse respiratory processes .

What is known about the genomic context of the dsy4157 gene?

The dsy4157 gene is part of the Desulfitobacterium hafniense DCB-2 genome, which consists of a 5,279,134-bp circular chromosome containing 5,042 predicted genes . While specific information about the genomic neighbors of dsy4157 is limited in the provided search results, the D. hafniense genome is known to contain numerous genes related to respiratory processes, including 53 molybdopterin-binding oxidoreductases and 19 flavoprotein paralogs of the fumarate reductase . The genomic context analysis would require examination of the regions flanking the dsy4157 gene to identify potential operonic structures or functional gene clusters.

What structural features characterize the DSY4157 protein?

The DSY4157 protein is classified as a UPF0060 family membrane protein . While detailed structural information is not explicitly provided in the search results, as a membrane protein, it likely contains hydrophobic domains that anchor it within the cell membrane. The full-length protein consists of 108 amino acids , suggesting it is a relatively small membrane protein. For comprehensive structural characterization, techniques such as the droplet-on-hydrogel bilayer approach mentioned in search result could potentially be applied to study its membrane integration and functional properties.

How does DSY4157 integrate into the complex respiratory network of D. hafniense?

D. hafniense possesses a remarkably versatile respiratory system, utilizing various electron acceptors including halogenated organic compounds and metals . While the specific role of DSY4157 in this respiratory network is not explicitly detailed in the search results, research on D. hafniense has revealed the importance of complex I-like enzymes in energy metabolism, particularly when lactate or pyruvate serves as electron donors .

To understand DSY4157's integration into this network, researchers should consider:

• Potential interactions with electron transport chain components

• Possible roles in membrane-associated respiratory complexes

• Connections to the organism's ability to use diverse electron acceptors

The inhibitor studies with rotenone described in search result demonstrate how the respiratory system in D. hafniense responds differently depending on electron donor types, suggesting a complex regulatory network that DSY4157 might participate in.

What methodologies can be employed to elucidate DSY4157's role in the electron transport chain?

To investigate DSY4157's potential role in electron transport:

• Inhibitor studies similar to those performed with rotenone on complex I-like enzymes

• Proteomic analyses under various growth conditions to identify changes in DSY4157 expression patterns

• Gene knockout/knockdown studies to observe phenotypic changes

• Protein-protein interaction studies to identify binding partners

The approach described in search result , where researchers calculated Euclidean distances between protein expression patterns to identify potential functional relationships, could be particularly valuable. This method revealed seven candidates with expression patterns similar to Nuo homologues in D. hafniense DCB-2 , and a similar approach might identify proteins functionally related to DSY4157.

How might post-translational modifications affect DSY4157 function in different metabolic states?

While the search results don't specifically address post-translational modifications (PTMs) of DSY4157, membrane proteins often undergo modifications that regulate their activity. Research approaches to investigate this question would include:

• Mass spectrometry analysis of purified DSY4157 under different growth conditions

• Phosphoproteomic studies to identify potential regulatory phosphorylation sites

• Investigation of other modifications like glycosylation or lipidation that might affect membrane localization

The proteomic dataset mentioned in search result could potentially be mined for evidence of PTMs on DSY4157 across different growth conditions.

What expression systems are optimal for recombinant production of DSY4157?

Based on the available information, E. coli has been successfully used as an expression host for recombinant production of His-tagged DSY4157 . When designing an expression system for this membrane protein, researchers should consider:

• Expression vector selection: Vectors with tunable expression control are preferable for membrane proteins to prevent toxic accumulation

• Fusion tags: The His-tag approach has been demonstrated , but alternative tags like MBP or SUMO might improve solubility

• Growth conditions: Lower temperatures (16-25°C) often improve proper folding of membrane proteins

• Membrane fraction isolation: Optimized protocols for membrane protein extraction from E. coli

For functional studies, the droplet-on-hydrogel bilayer technique described in search result offers "independent control of the electrical and chemical transmembrane potential," which could be valuable for characterizing DSY4157's membrane-associated functions.

How can researchers effectively study DSY4157 in the context of anaerobic respiration?

To study DSY4157 in anaerobic respiration contexts, researchers should implement methodologies similar to those described for D. hafniense cultivation and analysis :

• Anaerobic culture techniques:

  • Use of anaerobic flasks with appropriate basal medium

  • Careful selection of electron donors (pyruvate, lactate, H₂) and acceptors (fumarate, chlorinated compounds)

  • Specific media adjustments for different growth conditions

• Analytical approaches:

  • Growth monitoring via spectrophotometry (OD₆₀₀)

  • Specific inhibitor studies (e.g., rotenone, piericidin A) to probe respiratory chain components

  • Proteomic analysis across different growth conditions

• Comparative analysis:

  • Contrast DSY4157 expression and function between respiratory conditions (e.g., H₂/fumarate vs. lactate/fumarate)

  • Examine expression patterns in relation to known respiratory components

What purification strategies yield functional DSY4157 for in vitro studies?

For membrane protein purification, researchers should consider:

• Detergent screening to identify optimal solubilization conditions

• Affinity chromatography utilizing the His-tag

• Size exclusion chromatography for final purification and buffer exchange

• Lipid reconstitution methods for functional studies

The integrity of purified DSY4157 should be verified through:

• SDS-PAGE and western blotting

• Mass spectrometry

• Circular dichroism to assess secondary structure

• Functional assays appropriate to hypothesized activity

How does DSY4157 compare with homologous proteins in other bacterial species?

While the search results don't provide direct homology information for DSY4157, researchers can approach this question by:

• Conducting bioinformatic analyses:

  • BLAST searches against bacterial protein databases

  • Multiple sequence alignments to identify conserved domains

  • Phylogenetic analysis to determine evolutionary relationships

• Comparing expression patterns:

  • Examine if homologs in other species show similar regulation under comparable growth conditions

  • Determine if genomic context is conserved across species

The UPF0060 protein family designation suggests that DSY4157 belongs to a group of proteins with uncharacterized function, making comparative analyses particularly valuable for generating functional hypotheses.

What role might DSY4157 play in D. hafniense's remarkable metabolic versatility?

D. hafniense demonstrates exceptional metabolic versatility, including dehalogenation, metal reduction, N₂ and CO₂ fixation capabilities . To explore DSY4157's potential contribution:

• Analyze DSY4157 expression across diverse growth conditions:

  • Different electron donors (H₂, lactate, pyruvate)

  • Various electron acceptors (fumarate, chlorinated compounds, metals)

  • Autotrophic vs. heterotrophic growth

• Investigate potential involvement in:

  • Membrane-associated electron transport

  • Adaptation to changing redox conditions

  • Stress responses related to membrane integrity

The approach used in search result , calculating Z-scores of protein abundance across growth conditions and identifying proteins with similar expression patterns, would be valuable for placing DSY4157 in specific metabolic contexts.

How does DSY4157 function within D. hafniense's complex regulatory network?

D. hafniense DCB-2's genome encodes 43 RNA polymerase sigma factors (including 27 sigma-24 subunits) and 59 two-component signal transduction systems , suggesting sophisticated regulatory mechanisms. To investigate DSY4157's place in this network:

• Examine transcriptional regulation:

  • Identify promoter elements upstream of dsy4157

  • Determine which sigma factors might regulate its expression

  • Analyze expression correlation with known regulatory elements

• Study post-transcriptional regulation:

  • Investigate mRNA stability under different conditions

  • Examine potential sRNA regulators

• Analyze potential interactions with signaling systems:

  • Explore relationships with two-component systems

  • Investigate response to environmental signals relevant to membrane function

What are the main challenges in studying membrane proteins like DSY4157, and how can they be overcome?

Membrane proteins present several unique challenges:

• Expression challenges:

  • Toxicity during overexpression

  • Improper folding or aggregation

  • Solution: Use tunable expression systems, fusion partners, and lower expression temperatures

• Purification difficulties:

  • Finding appropriate detergents for solubilization

  • Maintaining protein stability outside native membrane

  • Solution: Systematic detergent screening, lipid supplementation during purification

• Functional characterization:

  • Reconstituting proper membrane environment

  • Solution: The droplet-on-hydrogel bilayer technique described in search result offers "a high-resolution functional study of membrane proteins with independent control of the electrical and chemical transmembrane potential"

How can researchers integrate omics data to develop hypotheses about DSY4157 function?

To leverage omics approaches:

• Integrate multiple data types:

  • Genomics: Analyze gene neighborhood and potential operonic structures

  • Transcriptomics: Examine expression patterns across conditions

  • Proteomics: Analyze protein abundance changes using approaches like the Z-score method described in search result

  • Metabolomics: Identify metabolic changes that correlate with DSY4157 expression

• Apply computational methods:

  • Correlation networks to identify functionally related genes/proteins

  • Pathway enrichment analysis

  • Protein-protein interaction predictions

• Validate computational predictions:

  • Targeted experiments based on hypotheses generated from omics data

  • Knockout/knockdown studies followed by omics analysis to observe system-wide effects