Recombinant UPF0176 protein VPA1597 (VPA1597)

What is UPF0176 protein VPA1597 and what are its key structural features?

UPF0176 protein VPA1597 is a bacterial protein characterized by a Rhodanese-like domain implicated in sulfur metabolism. Similar to other UPF0176 family members, it contains a conserved UPF0176 motif whose specific function remains largely unknown but is bacterial-specific. The protein features structural similarities to other members of the UPF0176 family, such as SPy_0915/M5005_Spy0717 from Streptococcus pyogenes, which contains the characteristic Rhodanese-like domain. The structure-function relationship of this protein remains an active area of research, particularly regarding its potential sulfurtransferase activity and role in bacterial metabolism.

Which expression systems are optimal for recombinant UPF0176 protein VPA1597 production?

Recombinant UPF0176 protein VPA1597 can be expressed and purified from multiple host systems, each offering specific advantages. E. coli and yeast expression systems typically provide the highest yields and shorter production timeframes, making them suitable for initial characterization studies . For applications requiring post-translational modifications essential for proper protein folding or activity, insect cells with baculovirus or mammalian expression systems are recommended despite their lower yields . The table below compares typical expression parameters based on data from related UPF0176 proteins:

What purification strategies are most effective for UPF0176 protein VPA1597?

The most effective purification strategy for UPF0176 protein VPA1597 typically involves a multi-step approach tailored to the expression system used. For His-tagged variants expressed in E. coli, immobilized metal affinity chromatography (IMAC) using Ni-NTA columns provides efficient initial purification. This should be followed by size exclusion chromatography to remove protein aggregates and achieve >85% purity. For mammalian cell-expressed protein, where native folding is prioritized, a combination of ion exchange chromatography and hydrophobic interaction chromatography often yields better results than affinity-based methods alone. Researchers should implement buffer optimization during purification, as the VPA1597 protein can exhibit variable stability depending on pH and salt concentration, similar to other Rhodanese-domain containing proteins.

How can I experimentally characterize the sulfurtransferase activity of UPF0176 protein VPA1597?

Characterizing the suspected sulfurtransferase activity of UPF0176 protein VPA1597 requires multiple complementary approaches. The primary method employs a thiosulfate:cyanide sulfurtransferase assay, measuring the formation of thiocyanate spectrophotometrically at 460 nm after reaction with ferric nitrate. Experimental design should include the following control conditions:

• Positive control using well-characterized rhodanese enzymes (e.g., bovine liver rhodanese)

• Negative control using heat-inactivated VPA1597

• Substrate specificity assessment using various sulfur donors (thiosulfate, mercaptopyruvate, etc.)

• Enzyme kinetics analysis to determine Km and Vmax values

Additionally, isothermal titration calorimetry (ITC) can provide thermodynamic parameters of substrate binding, while mutations of conserved active site residues can confirm the catalytic mechanism. Researchers should be aware that the activity might be metal-dependent, despite the lack of confirmed metal-binding sites in UPF0176 proteins. Thus, including EDTA controls and metal supplementation experiments is recommended for comprehensive activity characterization.

What approaches can resolve the discrepancy between in vitro solubility and in vivo localization of UPF0176 proteins?

The discrepancy between in vitro solubility and in vivo localization observed in UPF0176 family proteins, including VPA1597, presents a significant research challenge. To address this, researchers should implement a multi-faceted experimental design:

• Fluorescently-tagged protein expression in bacterial cells to track subcellular localization via confocal microscopy

• Fractionation studies comparing membrane-associated versus cytosolic protein fractions

• Crosslinking mass spectrometry to identify potential interaction partners that may affect localization

• In vitro lipid binding assays to assess potential membrane interactions

The experimental approach should systematically test hypotheses about localization determinants, including post-translational modifications, protein-protein interactions, and environmental conditions like pH or oxidative stress that might trigger conformational changes. These methods can help determine whether the observed discrepancy is a biological phenomenon or an artifact of experimental conditions, ultimately providing insights into the protein's native function and regulation.

How can structural dynamics data for UPF0176 protein VPA1597 be generated and analyzed?

Generating and analyzing structural dynamics data for UPF0176 protein VPA1597 requires an integrated approach combining experimental techniques and computational methods. While structural dynamics data is currently lacking for UPF0176 proteins, researchers can implement the following methodology:

Analysis should focus on correlating structural dynamics with functional hypotheses, particularly regarding the Rhodanese-like domain's potential catalytic mechanism and substrate binding-induced conformational changes. Researchers should also examine conserved residues across the UPF0176 family to identify functionally important regions for targeted mutagenesis studies.

What experimental controls are essential when studying UPF0176 protein VPA1597 interaction with potential binding partners?

When investigating UPF0176 protein VPA1597 interactions with potential binding partners, implementing rigorous controls is crucial for reliable results. The experimental design should include:

• Negative controls:

  • Non-specific proteins of similar size/charge to rule out non-specific binding

  • Tag-only controls when using tagged VPA1597 to distinguish tag-mediated interactions

  • Buffer-only controls to establish baseline readings

• Positive controls:

  • Known protein-protein interactions with similar binding affinities

  • Synthetic peptides derived from predicted binding interfaces

• Validation controls:

  • Concentration gradients to distinguish specific from non-specific interactions

  • Competition assays with unlabeled protein

  • Mutated variants of binding interfaces to confirm specificity

For protein-protein interaction studies using biotinylated variants of VPA1597, additional controls should address biotin interference and orientation constraints. Multiple complementary methods are recommended, including Surface Plasmon Resonance (SPR), co-immunoprecipitation, and proximity ligation assays. Researchers should also account for the lack of commercial antibodies against UPF0176 proteins by developing custom antibodies or using epitope tags that don't interfere with binding interactions.

How should researchers design experiments to characterize the substrate specificity of UPF0176 protein VPA1597?

Designing experiments to characterize the substrate specificity of UPF0176 protein VPA1597, which remains largely uncharacterized, requires a systematic approach:

• Initial substrate screening:

  • Test structurally diverse sulfur-containing compounds based on known rhodanese substrates

  • Include thiosulfate, mercaptopyruvate, lipoic acid, and various persulfides

  • Monitor activity using spectrophotometric assays or mass spectrometry

• Kinetic parameter determination:

  • Measure reaction rates across substrate concentration ranges to determine Km, Vmax, and catalytic efficiency (kcat/Km)

  • Compare kinetic parameters across substrates to identify preferences

• Structure-activity relationship analysis:

  • Test structural analogs of active substrates to identify essential chemical features

  • Use molecular docking to predict binding modes and guide analog design

• Physiologically relevant substrate identification:

  • Implement metabolomics approaches to identify endogenous substrates in bacterial lysates

  • Use activity-based protein profiling with sulfur-reactive probes

The experimental design should include proper negative controls (heat-inactivated enzyme, non-catalytic mutants) and positive controls (known rhodanese enzymes). Data analysis should carefully distinguish between primary and secondary enzyme activities, as rhodanese domain proteins often exhibit promiscuity toward structurally related compounds.

What are the critical considerations for designing site-directed mutagenesis experiments for UPF0176 protein VPA1597?

Site-directed mutagenesis experiments for UPF0176 protein VPA1597 require careful planning to ensure meaningful functional insights. Critical considerations include:

• Target residue selection:

  • Conserved residues across UPF0176 family members

  • Predicted catalytic residues in the Rhodanese-like domain

  • Residues at the conserved UPF0176 motif

  • Surface-exposed residues that might mediate protein-protein interactions

• Mutation design strategy:

  • Conservative substitutions (e.g., Asp to Glu) to test charge importance

  • Non-conservative substitutions (e.g., Cys to Ser) to test specific chemical properties

  • Alanine scanning of putative binding interfaces

  • Introduction of bulky residues to test spatial constraints

• Expression and stability controls:

  • Verify that mutations don't compromise protein folding using thermal shift assays

  • Confirm similar expression levels between wild-type and mutant proteins

  • Assess secondary structure integrity via circular dichroism spectroscopy

• Functional assays:

  • Compare catalytic parameters (kcat, Km) between wild-type and mutant proteins

  • Assess binding affinity changes for substrates or interaction partners

  • Evaluate changes in subcellular localization patterns

Researchers should design mutations based on available structural information or homology models if crystal structures are unavailable. For UPF0176 proteins, mutagenesis of conserved active site cysteines in the Rhodanese domain would be particularly informative for testing the proposed sulfurtransferase activity. Additionally, creating a comprehensive mutation library rather than testing isolated mutations provides more robust insights into structure-function relationships.

How can researchers address the lack of commercial antibodies for UPF0176 protein VPA1597?

The lack of commercial antibodies for UPF0176 proteins presents a significant research challenge. Researchers can implement several strategies to overcome this limitation:

• Custom antibody development:

  • Generate recombinant protein or synthetic peptides corresponding to unique epitopes of VPA1597

  • Produce polyclonal antibodies in rabbits or chickens for broader epitope recognition

  • Develop monoclonal antibodies targeting specific domains for higher specificity

  • Validate antibody specificity using knockout controls or competing peptides

• Alternative detection strategies:

  • Epitope tagging: Introduce small tags (FLAG, HA, His6) that don't interfere with function

  • Split-GFP complementation for protein interaction studies

  • Proximity labeling approaches (BioID, APEX) to identify interacting proteins

  • Mass spectrometry-based protein identification from pulldowns or immunoprecipitations

• Antibody-free detection methods:

  • Aptamer development through SELEX (Systematic Evolution of Ligands by Exponential Enrichment)

  • Nanobodies derived from camelid antibodies, which offer smaller size and higher stability

  • Label-free detection technologies like surface plasmon resonance

When generating custom antibodies, researchers should target unique regions of VPA1597 rather than the highly conserved Rhodanese domain to minimize cross-reactivity with related proteins. The developed reagents should undergo rigorous validation including western blotting, immunoprecipitation, and immunofluorescence to ensure specificity and sensitivity across multiple applications.

What computational approaches can help predict UPF0176 protein VPA1597 function in the absence of comprehensive experimental data?

In the absence of comprehensive experimental data, several computational approaches can aid in predicting UPF0176 protein VPA1597 function:

• Homology-based predictions:

  • Sequence alignment with characterized proteins sharing the Rhodanese domain

  • Domain architecture analysis to identify functional motifs

  • Phylogenetic profiling to identify co-evolved genes suggesting functional relationships

• Structure-based predictions:

  • Homology modeling based on related structures

  • Molecular docking with potential substrates

  • Binding site prediction using CASTp, COACH, or similar algorithms

  • Electrostatic surface analysis to identify potential interaction interfaces

• Network-based predictions:

  • Gene neighborhood analysis in bacterial genomes

  • Co-expression pattern analysis across various conditions

  • Protein-protein interaction network prediction using interolog mapping

• Machine learning approaches:

  • Function prediction using tools like DeepFRI or COFACTOR

  • Substrate specificity prediction using enzyme fingerprinting methods

  • Cellular localization prediction using signal sequence analyzers

The integration of multiple computational methods often provides more reliable predictions than any single approach. Researchers should prioritize hypotheses that are supported by multiple computational methods and design targeted experiments to validate these predictions. For UPF0176 proteins, particular attention should be paid to predictions related to sulfur metabolism pathways due to the presence of the Rhodanese-like domain.

How can researchers resolve conflicting data about metal-binding properties of UPF0176 family proteins?

The conflicting data regarding metal-binding properties of UPF0176 family proteins, including the lack of confirmed metal-binding sites despite conserved motifs, can be addressed through a comprehensive experimental strategy:

• Metal binding characterization:

  • Inductively coupled plasma mass spectrometry (ICP-MS) to identify bound metals in purified protein

  • Isothermal titration calorimetry (ITC) to measure binding affinities for various metals

  • Differential scanning fluorimetry to assess thermal stability changes upon metal binding

  • Metal-catalyzed oxidation (MCO) assays to identify metal-binding sites

• Structural studies:

  • X-ray absorption spectroscopy (XAS) to characterize metal coordination environment

  • Crystallography with anomalous scattering to definitively locate metal ions

  • NMR studies with paramagnetic metals to identify binding sites

• Functional validation:

  • Activity assays in the presence of metal chelators (EDTA, EGTA)

  • Metal reconstitution experiments with various metals to restore activity

  • Site-directed mutagenesis of predicted metal-coordinating residues

• Data integration:

  • Meta-analysis of published data on related proteins

  • Computational modeling of metal binding sites based on conserved motifs

  • Correlation of metal binding with environmental or pathogenic contexts

Researchers should be particularly careful about metal contamination during protein purification, which can lead to conflicting results. Experiments should include appropriate negative controls (metal-free conditions) and positive controls (proteins with well-characterized metal binding). When analyzing results, considerations should include the possibility that metal binding might be conditional, depending on protein conformation, redox state, or the presence of specific substrates or protein partners.