Recombinant Synechocystis sp. UPF0093 membrane protein slr1790 (slr1790)

What is UPF0093 membrane protein slr1790 and what organism does it originate from?

The UPF0093 membrane protein slr1790 is an integral membrane protein encoded by the slr1790 gene in the cyanobacterium Synechocystis sp. strain PCC 6803/Kazusa. This protein belongs to the UPF0093 family of uncharacterized proteins with predicted membrane-spanning domains. The complete amino acid sequence consists of 210 amino acids with multiple predicted transmembrane helices . Synechocystis sp. PCC 6803 is a widely studied model cyanobacterium known for its ability to perform photosynthesis and its completely sequenced genome, making it valuable for membrane protein research .

How does slr1790 compare to other membrane proteins in Synechocystis sp. PCC 6803?

Synechocystis sp. PCC 6803 contains approximately 60 experimentally identified integral membrane proteins with diverse functions . The plasma membrane proteome includes proteins involved in nutrient uptake, secretion, chemotaxis, and energy transduction. Unlike many identified membrane proteins with known functions (such as photosynthetic complexes or transporters), slr1790 belongs to the UPF (uncharacterized protein family) classification, indicating its function remains to be fully elucidated. The pI distribution of Synechocystis membrane proteins varies significantly, with both acidic and basic proteins represented in the membrane proteome .

What are the recommended expression systems for recombinant production of slr1790?

For recombinant expression of slr1790, several systems can be considered:

• Homologous expression in Synechocystis: This approach maintains the native cellular environment and post-translational modifications but typically yields lower protein quantities.

• E. coli-based systems: These offer high yields but may require optimization for membrane protein expression:

  • C41(DE3) or C43(DE3) strains specifically developed for membrane protein expression

  • pET-based vectors with tunable promoters to control expression rates

  • Fusion tags (such as MBP or SUMO) to enhance solubility

• Cell-free expression systems: These avoid toxicity issues often encountered with membrane protein overexpression.

When designing expression constructs, researchers should consider including purification tags (His, FLAG, or Strep) positioned to avoid interference with membrane insertion. Expression should be optimized by testing various induction conditions (temperature, inducer concentration, and induction time) .

What purification strategies are most effective for isolating recombinant slr1790?

Purification of membrane proteins like slr1790 requires specialized approaches:

• Membrane fraction isolation:

  • Cell disruption by sonication or French press

  • Differential centrifugation to isolate membrane fractions

  • Urea washing (as employed in Synechocystis membrane studies) to remove peripheral proteins

• Solubilization:

  • Test multiple detergents (DDM, LDAO, or CHAPS) at various concentrations

  • Consider alternative solubilization with styrene-maleic acid copolymers (SMAs) to maintain lipid environment

• Purification methods:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Size exclusion chromatography for further purification and buffer exchange

  • Ion exchange chromatography as a complementary step

The purification protocol should be optimized to maintain protein stability, as detergent selection significantly impacts membrane protein integrity during isolation .

How can researchers verify the structural integrity of purified slr1790?

Verification of structural integrity is critical for membrane protein studies:

• Biophysical techniques:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Fluorescence spectroscopy to monitor tertiary structure

  • Dynamic light scattering (DLS) to verify homogeneity and detect aggregation

• Functional assays:

  • Binding assays if ligands are identified

  • Reconstitution into liposomes to test functionality

• Structural analysis:

  • Negative-stain electron microscopy for initial structural assessment

  • Blue native PAGE to analyze oligomeric state and complex formation

Each technique provides complementary information about protein folding and stability, which is particularly important for membrane proteins that often denature during purification .

What approaches can be used to study potential interaction partners of slr1790?

Several complementary approaches can identify interaction partners:

• Co-immunoprecipitation (Co-IP):

  • Generate antibodies against slr1790 or use epitope tags

  • Perform pull-downs from solubilized membranes

  • Identify co-precipitating proteins by mass spectrometry

• Crosslinking-mass spectrometry (XL-MS):

  • Apply chemical crosslinkers to stabilize transient interactions

  • Digest and analyze crosslinked peptides by mass spectrometry

  • This approach has proven valuable for cyanobacterial protein complex studies

• Membrane-based interaction screens:

  • Vesicle-based membrane protein display platforms allow for unbiased detection of interactions

  • This approach is particularly valuable for maintaining membrane proteins in their native conformation

• Split reporter assays:

  • Bacterial two-hybrid or split-GFP systems adapted for membrane proteins

  • Allows in vivo detection of protein-protein interactions

A combination of these methods provides the most comprehensive view of the interactome .

How can transcriptomic and proteomic data be used to infer the function of slr1790?

Integrative omics approaches offer valuable insights into protein function:

The combination of these approaches can provide hypotheses about function that can be experimentally tested .

What structural analysis methods are most suitable for characterizing membrane proteins like slr1790?

Several structural biology techniques are applicable to membrane proteins:

• X-ray crystallography:

  • Requires highly pure, stable, and crystallizable protein

  • Often requires extensive construct optimization and crystallization screening

  • Provides high-resolution structural information

• Cryo-electron microscopy (cryo-EM):

  • Single-particle analysis for larger proteins or complexes

  • Does not require crystallization

  • Resolution has improved significantly in recent years

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Solution NMR for smaller membrane proteins or domains

  • Solid-state NMR for larger membrane proteins

  • Provides dynamic information not available from static structures

• Molecular dynamics simulations:

  • Computational approach to model protein behavior in membranes

  • Can predict conformational changes and lipid interactions

  • Requires experimental validation

The choice of method depends on protein size, stability, and research questions, with a multi-technique approach often providing the most comprehensive structural insights .

What are the critical parameters for maintaining stability of slr1790 during experimental procedures?

Maintaining membrane protein stability requires attention to several parameters:

• Buffer composition:

  • pH: Test range 6.5-8.0 (physiological for Synechocystis)

  • Salt concentration: Typically 100-300 mM to maintain ionic strength

  • Glycerol (10-20%): Acts as a stabilizing agent

  • Reducing agents (DTT or β-mercaptoethanol) to prevent oxidation

• Detergent considerations:

  • Concentration: Maintain above critical micelle concentration (CMC)

  • Detergent type: Milder detergents (DDM, LMNG) better preserve structure

  • Consider detergent exchange during purification

• Temperature management:

  • Perform all manipulations at 4°C when possible

  • Avoid freeze-thaw cycles (aliquot and store at -80°C)

  • Consider storage in 50% glycerol for extended stability

• Handling precautions:

  • Minimize exposure to air/oxygen

  • Use silanized glass or low-binding plastic to prevent adsorption

  • Avoid vortexing (use gentle mixing)

Careful optimization of these parameters is essential for maintaining functional integrity throughout experimental procedures .

How can researchers troubleshoot expression problems with recombinant slr1790?

Common expression issues and solutions include:

• Low expression levels:

  • Optimize codon usage for the expression host

  • Test different promoter strengths

  • Reduce expression temperature (16-20°C)

  • Use specialized strains (C41/C43, Rosetta)

• Protein toxicity:

  • Use tightly controlled inducible systems

  • Balance expression level through inducer concentration

  • Consider cell-free expression systems

• Inclusion body formation:

  • Lower induction temperature and inducer concentration

  • Add fusion partners (MBP, SUMO, Trx)

  • Test different detergents for solubilization

  • Develop refolding protocols if necessary

• Degradation issues:

  • Add protease inhibitors during all steps

  • Test expression in protease-deficient strains

  • Optimize purification speed to minimize exposure time

• Verification methods:

  • Western blotting with antibodies against the protein or tag

  • Mass spectrometry for protein identification

  • Functional assays to confirm proper folding

Systematic troubleshooting of these parameters is often necessary to achieve successful expression .

What controls should be included in slr1790 functional and interaction studies?

Robust experimental design requires appropriate controls:

• Negative controls:

  • Empty vector/untransformed cells for expression studies

  • Unrelated membrane protein of similar size for specificity testing

  • Detergent-only samples for binding studies

  • Non-specific antibodies in immunoprecipitation experiments

• Positive controls:

  • Well-characterized membrane protein from Synechocystis

  • Known protein-protein interactions in the same experimental system

  • Validated binding partners if available

• Technical validation:

  • Multiple biological and technical replicates

  • Alternative methods to confirm key findings

  • Dose-response relationships for interaction studies

  • Competition experiments to confirm specificity

• Complementary approaches:

  • In vivo validation of in vitro findings

  • Genetic approaches (knockouts, complementation)

  • Computational predictions with experimental validation

Proper controls distinguish genuine interactions from technical artifacts, particularly important for membrane proteins that often exhibit non-specific interactions .

How should contradictory results in slr1790 functional studies be interpreted?

Contradictory results are common in membrane protein research and should be approached systematically:

• Methodological differences:

  • Compare experimental conditions (detergents, buffers, temperature)

  • Assess protein preparation methods (tags, constructs, purification)

  • Consider differences in analytical techniques

• Biological variables:

  • Growth conditions of Synechocystis (light intensity, media composition)

  • Genetic background differences between strains

  • Physiological state of cells (exponential vs. stationary phase)

• Resolution strategies:

  • Perform side-by-side comparisons under identical conditions

  • Collaborate with labs reporting contradictory results

  • Use orthogonal approaches to test the same hypothesis

  • Consider post-translational modifications or conformational states

• Interpretation framework:

  • Develop models that accommodate seemingly contradictory data

  • Consider context-dependent functions

  • Evaluate statistical significance and biological relevance

Scientific progress often emerges from resolving contradictions through careful experimental design and open scientific discourse .

What bioinformatics tools are most useful for analyzing slr1790 and related proteins?

Several bioinformatics tools provide valuable insights for membrane protein analysis:

• Sequence analysis:

  • TMHMM/HMMTOP: Transmembrane helix prediction

  • SignalP: Signal peptide identification

  • PSIPRED: Secondary structure prediction

  • ConSurf: Evolutionary conservation mapping

• Structural prediction:

  • AlphaFold2: De novo structure prediction

  • I-TASSER: Template-based modeling

  • SWISS-MODEL: Homology modeling

  • PredictProtein: Integrated structure/function prediction

• Functional prediction:

  • InterPro: Protein family and domain recognition

  • STRING: Protein-protein interaction networks

  • KEGG: Pathway mapping and analysis

  • CyanoBase: Cyanobacteria-specific genomic database

• Data integration platforms:

  • UniProt: Comprehensive protein information

  • RCSB PDB: Structural database

  • Membrane Protein Data Bank: Specialized for membrane proteins

These tools should be used in combination to build a comprehensive understanding of protein features and potential functions .

How can transcriptomic data be leveraged to understand slr1790 regulation?

RNA-seq and other transcriptomic data provide valuable insights into regulation:

• Expression pattern analysis:

  • Compare slr1790 expression across different growth conditions

  • Identify conditions that induce or repress expression

  • RNA-seq studies in Synechocystis have identified genes regulated under different nutrient conditions

• Co-expression networks:

  • Identify genes with similar expression patterns

  • Construct networks of co-regulated genes

  • Infer potential functional relationships

• Response to environmental factors:

  • Light conditions (intensity, quality)

  • Nutrient availability (nitrogen, phosphorus)

  • Stress responses (oxidative, temperature)

  • Under nitrogen-deficient conditions, Synechocystis shows significant transcriptional remodeling

• Regulatory element identification:

  • Promoter analysis for transcription factor binding sites

  • Identification of potential regulatory RNAs

  • Comparative genomics to identify conserved regulatory elements

Analysis of existing datasets from experiments with Synechocystis under various conditions can provide hypotheses about slr1790 regulation .