Recombinant Synechocystis sp. UPF0150 protein ssr1765 (ssr1765)

What is the UPF0150 domain found in the ssr1765 protein?

The UPF0150 domain (IPR005357) represents a protein domain of unknown function that appears in several proteins across cyanobacterial species, including ssr1765 in Synechocystis sp. PCC6803. In large-scale protein-protein interaction studies, this domain has been occasionally observed among proteins that exhibited self-activation in yeast two-hybrid assays . The domain's precise function remains undetermined, but its conservation across species suggests biological significance. Structural prediction analyses indicate potential involvement in signaling or regulatory functions, possibly through protein-protein interactions that modulate cellular responses to environmental conditions.

How is the ssr1765 gene organized in the Synechocystis genome?

The ssr1765 gene is located on the chromosome of Synechocystis sp. PCC6803, which was the first phototrophic organism to have its genome completely sequenced . The gene encodes a protein containing the UPF0150 domain. When examining the genomic context, researchers should analyze adjacent genes that might form an operon with ssr1765, as this could provide functional insights. Comparative genomics approaches using tools like CyanoBase can reveal conserved gene neighborhoods that might suggest functional relationships with other genes in signaling or metabolic pathways.

What protein-protein interactions have been identified for ssr1765?

Large-scale protein-protein interaction analyses in Synechocystis have identified multiple potential interaction partners for proteins containing domains like UPF0150. Using modified high-throughput yeast two-hybrid assays, researchers have screened genes including those of unknown function such as ssr1765 . When investigating ssr1765 interactions, researchers should evaluate each interaction using the interaction generality (IG) measure to assess the reliability of detected interactions. Lower IG values (closer to 1) generally indicate more reliable and specific interactions, while higher values may represent promiscuous interactions that require additional validation through complementary methods.

What expression systems are optimal for recombinant ssr1765 production?

Based on successful recombinant protein expression of other Synechocystis proteins, E. coli represents a viable expression system for ssr1765 . For optimal expression, consider the following approach:

• Clone the ssr1765 gene into an expression vector containing an affinity tag (His-tag or GST)

• Transform into an E. coli strain optimized for protein expression (BL21(DE3), Rosetta, or Arctic Express)

• Conduct small-scale expression trials testing different conditions:

  • Induction temperatures (16°C, 25°C, 37°C)

  • IPTG concentrations (0.1-1.0 mM)

  • Expression duration (4-24 hours)

For proteins that demonstrate self-activation properties like some UPF0150 domain-containing proteins, maintaining proper solubility and preventing aggregation is crucial. Consider testing different buffer conditions and additives (glycerol, low concentrations of detergents) during purification to maintain protein stability and solubility.

How can yeast two-hybrid assays be optimized for studying ssr1765 interactions?

When designing yeast two-hybrid assays for ssr1765, researchers should address potential self-activation issues common to UPF0150 domain-containing proteins by:

• Performing thorough self-activation tests before screening using 3-AT (3-amino-1,2,4-triazole) titration

• Using multiple reporter genes driven by different GAL4-responsive promoters

• Creating truncated versions of ssr1765 that might reduce self-activation while maintaining interaction capacity

• Employing a modified yeast two-hybrid approach similar to the one described for Synechocystis proteins

Table 1: Optimization Parameters for Yeast Two-Hybrid Screening with ssr1765

What purification strategies yield highest purity recombinant ssr1765?

For purification of recombinant ssr1765 protein, a multi-step approach is recommended:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged protein)

• Intermediate purification via ion-exchange chromatography

• Final polishing step using size-exclusion chromatography

Based on observations from other Synechocystis proteins, recombinant proteins can form aggregates under low salt conditions . Therefore, maintain sufficient ionic strength (150-300 mM NaCl) in all buffers to prevent aggregation. Additionally, include 5-10% glycerol in buffers to improve protein stability. The addition of reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) may also enhance protein solubility by preventing disulfide bond formation.

How does the UPF0150 domain contribute to protein-protein interactions?

The UPF0150 domain in ssr1765 may play a significant role in mediating protein-protein interactions in Synechocystis signaling networks. Large-scale protein-protein interaction studies have shown that domains of unknown function, including UPF0150, were occasionally observed among proteins showing self-activation in yeast two-hybrid assays . This suggests the domain may have inherent interaction properties.

To investigate the specific contribution of the UPF0150 domain:

• Perform domain deletion and mutation analyses to identify critical residues for interaction

• Utilize complementary interaction detection methods (pull-down assays, co-immunoprecipitation, FRET)

• Compare interaction profiles between wild-type ssr1765 and domain mutants

• Conduct crosslinking studies coupled with mass spectrometry to identify precise interaction interfaces

The domain may function as a scaffold for assembling multi-protein complexes or as a regulatory module that modulates interaction specificity in response to environmental stimuli.

What is the relationship between ssr1765 and two-component signaling systems in Synechocystis?

Synechocystis contains 44 genes for histidine kinases (Hiks) and 42 genes for response regulators (Rres) that form the backbone of two-component signal transduction systems . While direct evidence linking ssr1765 to these systems is limited, protein-protein interaction studies may reveal connections.

To investigate potential roles of ssr1765 in two-component signaling:

• Screen for direct interactions between ssr1765 and known components of two-component systems

• Analyze phenotypes of ssr1765 deletion mutants under conditions known to activate specific two-component pathways

• Perform phosphorylation assays to determine if ssr1765 influences the phosphorylation state of response regulators

• Utilize phosphoproteomics to identify changes in the phosphorylation profile of cells lacking ssr1765

Understanding these relationships could provide insights into novel regulatory mechanisms in cyanobacterial signal transduction networks.

How does ssr1765 function change under different environmental conditions?

As a protein potentially involved in signaling networks, ssr1765 function may vary under different environmental conditions. To study these changes:

• Monitor expression levels of ssr1765 under various stress conditions (nutrient limitation, temperature shifts, light intensity changes)

• Analyze the interaction network of ssr1765 under different environmental conditions using quantitative proteomics

• Perform phenotypic characterization of ssr1765 deletion mutants under various growth conditions

• Conduct cellular localization studies to determine if ssr1765 localization changes in response to environmental cues

Understanding condition-dependent function changes may reveal the physiological role of this protein in cyanobacterial adaptation to environmental fluctuations.

What computational approaches can predict potential functions of ssr1765?

Multiple bioinformatic approaches can shed light on the potential functions of ssr1765:

• Sequence-based analyses:

  • Multiple sequence alignment of UPF0150 domain-containing proteins across species

  • Identification of conserved motifs and residues

  • Phylogenetic analysis to understand evolutionary relationships

• Structure-based predictions:

  • Secondary and tertiary structure prediction using tools like Phyre2, I-TASSER

  • Identification of structural homologs through fold recognition

  • Molecular docking to predict potential interaction partners

• Network-based approaches:

  • Integration of protein-protein interaction data from yeast two-hybrid studies

  • Construction of functional association networks using tools like STRING

  • Guilt-by-association analysis to predict function based on interaction partners

• Gene neighborhood analysis:

  • Examination of genomic context and gene clusters across cyanobacterial species

  • Identification of conserved gene neighborhoods that may suggest functional relationships

How can interaction data for ssr1765 be validated computationally?

Computational validation of protein-protein interaction data for ssr1765 can help prioritize experimental validation efforts:

• Calculate Interaction Generality (IG) values for each interaction, as described in previous studies

• Perform paralogue verification by checking if paralogues of interaction partners also interact with ssr1765

• Compare ssr1765 interaction data with interaction data from other organisms for orthologous proteins

• Analyze structural compatibility between ssr1765 and proposed interaction partners

• Evaluate co-expression patterns between ssr1765 and interaction partners across different conditions

Table 2: Computational Methods for Validating ssr1765 Interactions

How can self-activation issues be overcome when studying ssr1765 in yeast two-hybrid systems?

Self-activation, which has been observed with UPF0150 domain-containing proteins in yeast two-hybrid systems , presents a significant challenge when studying ssr1765. Researchers can employ several strategies to address this issue:

• Titrate 3-AT concentrations (from 0 to 10 mM) to determine the minimum concentration needed to suppress self-activation

• Create truncated versions of ssr1765 that retain functional domains but show reduced self-activation

• Use alternative yeast strains with different reporter gene configurations

• Employ modified yeast two-hybrid systems like the reverse two-hybrid system or split-ubiquitin system

• Validate interactions using complementary methods such as pull-down assays or FRET

For proteins showing strong self-activation that cannot be suppressed even with 10 mM 3-AT, consider alternative protein-protein interaction detection methods entirely.

How can protein aggregation be minimized during recombinant ssr1765 purification?

Based on observations from other Synechocystis proteins , researchers should consider the following approaches to minimize aggregation of recombinant ssr1765:

• Buffer optimization:

  • Maintain salt concentration between 150-300 mM NaCl

  • Include stabilizing agents like 5-10% glycerol

  • Add mild detergents (0.01-0.05% Tween-20 or 0.1% Triton X-100) if necessary

• Expression conditions:

  • Lower induction temperature (16-20°C)

  • Reduce IPTG concentration (0.1-0.25 mM)

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

• Protein engineering:

  • Remove hydrophobic patches identified through computational analysis

  • Create fusion constructs with solubility-enhancing tags (SUMO, MBP, TRX)

  • Consider surface entropy reduction mutagenesis to improve solubility

• Chromatographic techniques:

  • Use size-exclusion chromatography to separate aggregated from properly folded protein

  • Consider on-column refolding procedures if inclusion bodies form

What are promising approaches for determining the physiological role of ssr1765?

To elucidate the physiological function of ssr1765, several complementary approaches should be considered:

• Generation and phenotypic characterization of ssr1765 deletion and overexpression strains across various growth conditions

• Transcriptomics and proteomics comparisons between wild-type and ssr1765 mutant strains

• Metabolic profiling to identify pathways affected by ssr1765 mutation

• Subcellular localization studies using fluorescent protein fusions

• ChIP-seq or similar techniques to identify potential DNA-binding activities if nuclear localization is observed

• Interactome mapping under different physiological conditions using quantitative proteomics

Combining these approaches can provide a comprehensive understanding of ssr1765 function in the context of Synechocystis physiology.

How might structural studies advance our understanding of ssr1765 function?

Structural studies of ssr1765 would significantly enhance our understanding of its function:

The purity and solubility achievable with recombinant Synechocystis proteins expressed in E. coli suggest that ssr1765 could be amenable to crystallographic studies, potentially revealing structural features that underlie its function.