Recombinant Synechococcus elongatus UPF0754 membrane protein Synpcc7942_1098 (Synpcc7942_1098)

What is the UPF0754 membrane protein Synpcc7942_1098 and why is it significant for research?

The UPF0754 membrane protein Synpcc7942_1098 is a membrane-associated protein found in the cyanobacterium Synechococcus elongatus strain PCC 7942 (formerly known as Anacystis nidulans R2). This protein belongs to the UPF (Uncharacterized Protein Family) classification, specifically UPF0754, indicating that its precise biological function remains to be fully elucidated. The protein consists of 412 amino acids and has been assigned the UniProt accession number Q31P91 .

The significance of this protein lies in several areas. First, as a membrane protein in a photosynthetic cyanobacterium, it may play roles in cellular processes critical to photosynthesis, nutrient transport, or environmental sensing. Second, studying uncharacterized proteins like Synpcc7942_1098 contributes to our understanding of cyanobacterial membrane biology and potentially reveals novel functional mechanisms. Third, Synechococcus elongatus PCC 7942 serves as a model organism for studying photosynthesis and circadian rhythms, making its component proteins valuable research targets for understanding these fundamental biological processes.

How should the recombinant Synpcc7942_1098 protein be stored to maintain optimal activity?

For optimal storage of the recombinant Synechococcus elongatus UPF0754 membrane protein Synpcc7942_1098, the following conditions are recommended:

• Short-term storage: Store working aliquots at 4°C for up to one week .

• Medium-term storage: Store at -20°C in a buffer containing 50% glycerol .

• Long-term storage: Store at -80°C in small aliquots to minimize freeze-thaw cycles .

The protein is typically supplied in a Tris-based buffer containing 50% glycerol, which has been optimized for this specific protein . It is crucial to avoid repeated freeze-thaw cycles as these can lead to protein denaturation and loss of structural integrity . For experimental work requiring multiple uses, it is advisable to prepare small working aliquots from the stock solution and store them separately to minimize the need for repeated thawing of the entire stock.

What expression systems are most effective for producing recombinant Synpcc7942_1098 protein?

Based on available information, Escherichia coli serves as an effective heterologous expression system for producing recombinant Synpcc7942_1098 protein . The E. coli expression system offers several advantages for membrane protein production, including rapid growth, high protein yields, and established protocols for induction and extraction.

For optimal expression, consider the following methodological approaches:

• Expression vector selection: Vectors containing strong inducible promoters (T7, tac) are typically recommended for membrane protein expression. The choice between N-terminal or C-terminal tags should be determined based on the specific experimental requirements.

• E. coli strain optimization: Specialized E. coli strains such as C41(DE3), C43(DE3), or Rosetta(DE3) often yield better results for membrane protein expression compared to standard BL21(DE3) strains. These strains are engineered to better tolerate the toxic effects often associated with overexpressing membrane proteins.

• Induction conditions: Lower induction temperatures (16-25°C) and reduced IPTG concentrations (0.1-0.5 mM) typically produce better results for membrane proteins than standard conditions, as they slow down protein production and allow more time for proper membrane insertion.

• Co-expression with chaperones: Co-expressing molecular chaperones (GroEL/GroES, DnaK/DnaJ) can significantly improve the folding and stability of membrane proteins during expression.

The specific conditions for optimal expression of Synpcc7942_1098 would need to be empirically determined for each laboratory setting through expression trials and optimization experiments.

What are the most effective techniques for purifying the recombinant Synpcc7942_1098 protein while maintaining its native conformation?

Purification of membrane proteins like Synpcc7942_1098 presents unique challenges due to their hydrophobic nature and requirement for membrane-mimetic environments. A multi-step purification strategy is typically recommended:

• Membrane extraction: Following cell lysis, the membrane fraction containing the overexpressed protein should be isolated through differential centrifugation. The membrane proteins can then be solubilized using appropriate detergents.

• Detergent selection: For initial solubilization, mild detergents such as n-dodecyl-β-D-maltoside (DDM), n-decyl-β-D-maltoside (DM), or digitonin are recommended as they effectively solubilize membrane proteins while preserving native protein structure. A detergent screen may be necessary to identify the optimal detergent for Synpcc7942_1098.

• Affinity chromatography: The recombinant protein is typically expressed with an affinity tag to facilitate purification. Common tags include polyhistidine (His6), FLAG, or Strep-tag II. The choice of tag should consider both purification efficiency and potential effects on protein function.

• Size exclusion chromatography: Following affinity purification, size exclusion chromatography provides further purification and allows assessment of protein homogeneity and oligomeric state.

• Quality assessment: The purified protein should be assessed for purity using SDS-PAGE and for structural integrity using circular dichroism (CD) spectroscopy or fluorescence-based thermal stability assays.

A typical purification workflow might yield the following results:

How can researchers verify the correct folding and functionality of purified Synpcc7942_1098 protein?

Verifying the proper folding and functionality of membrane proteins like Synpcc7942_1098 requires multiple complementary approaches:

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure content and thermal stability

  • Fluorescence spectroscopy to evaluate tertiary structure through intrinsic tryptophan fluorescence

  • Dynamic light scattering (DLS) to confirm monodispersity and absence of aggregation

  • Thermal shift assays to assess protein stability in different buffer conditions

• Structural integrity assessment:

  • Limited proteolysis to probe for well-folded domains resistant to proteolytic digestion

  • Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to determine oligomeric state

  • Small-angle X-ray scattering (SAXS) for low-resolution structural information

• Functional assays:

  • As the specific function of UPF0754 proteins is not well-characterized, functional assays would need to be developed based on hypothesized functions

  • For membrane proteins, reconstitution into proteoliposomes or nanodiscs followed by functional assays often provides insights into transport or signaling activities

  • Binding assays with potential ligands or interaction partners can provide indirect evidence of proper folding

When working with proteins of unknown function like Synpcc7942_1098, comparative analysis with related proteins or homologs from other species can provide valuable insights into potential functional assays. Additionally, computational predictions of protein function based on structural features can guide the design of appropriate functionality tests.

What structural analysis techniques are most appropriate for characterizing the Synpcc7942_1098 membrane protein?

Due to the challenges associated with membrane protein structural analysis, a multi-technique approach is recommended for characterizing Synpcc7942_1098:

• X-ray crystallography: While challenging for membrane proteins, X-ray crystallography remains the gold standard for high-resolution structural determination. For Synpcc7942_1098, lipidic cubic phase (LCP) or bicelle crystallization methods may be more successful than traditional vapor diffusion techniques. Screening with various detergents, lipids, and stabilizing agents is crucial for crystal formation.

• Cryo-electron microscopy (cryo-EM): Recent advances in cryo-EM have revolutionized membrane protein structural biology. For Synpcc7942_1098, single-particle cryo-EM could be particularly valuable if the protein forms oligomers or complexes of sufficient size (>100 kDa). Smaller proteins may benefit from advances in microcrystal electron diffraction (MicroED).

• Nuclear magnetic resonance (NMR) spectroscopy: For specific domains or regions of Synpcc7942_1098, solution or solid-state NMR can provide valuable structural and dynamic information. This approach is particularly useful for studying protein-ligand interactions and conformational changes.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique provides information about protein dynamics and solvent accessibility, which can be particularly valuable for mapping functional regions or conformational changes in response to specific conditions.

• Computational structure prediction: With recent advances in AI-based structure prediction algorithms like AlphaFold2, computational approaches can provide valuable starting models for membrane proteins. These predictions can guide experimental design and interpretation of low-resolution structural data.

The choice of technique should be guided by the specific research questions being addressed. A comprehensive structural characterization would likely employ multiple complementary approaches.

How does the Synpcc7942_1098 protein compare to other membrane proteins in cyanobacteria, and what evolutionary insights can be gained?

A comparative analysis of Synpcc7942_1098 with other cyanobacterial membrane proteins provides valuable evolutionary insights:

• Homology analysis: The UPF0754 family includes membrane proteins found across various cyanobacterial species. Sequence analysis reveals that Synpcc7942_1098 shares significant homology with proteins in related cyanobacteria, suggesting conservation of function. The degree of sequence conservation varies across different regions of the protein, with certain domains showing higher conservation, potentially indicating functionally important regions.

• Phylogenetic distribution: The presence of UPF0754 family proteins across diverse cyanobacterial lineages suggests an ancient evolutionary origin, potentially dating back to early cyanobacterial evolution. Comparative genomic analysis can reveal whether this protein family is exclusive to cyanobacteria or has homologs in other bacterial phyla.

• Structural conservation: While specific structural data for Synpcc7942_1098 is limited, predictive modeling and comparison with structurally characterized membrane proteins can reveal conserved structural motifs. These may include transmembrane helical arrangements or loop regions involved in specific functions.

• Genomic context: Analyzing the genomic neighborhood of the Synpcc7942_1098 gene (Synpcc7942_1098) can provide insights into potential functional associations through gene clustering and operonic arrangements. In cyanobacteria, functionally related genes are often clustered together or co-regulated.

A detailed evolutionary analysis would include:

• Multiple sequence alignment of UPF0754 family proteins across diverse cyanobacterial species

• Identification of conserved sequence motifs and potential functional domains

• Reconstruction of phylogenetic relationships to track the evolution of this protein family

• Correlation of sequence/structural features with cyanobacterial adaptation to different ecological niches

What experimental approaches can be used to investigate the potential role of Synpcc7942_1098 in phosphate sensing or regulation?

Based on the presence of phosphate-related regulatory systems in Synechococcus elongatus PCC 7942, experimental approaches to investigate potential roles of Synpcc7942_1098 in phosphate metabolism could include:

• Gene knockout and phenotypic analysis:

  • Generate a Synpcc7942_1098 deletion mutant in Synechococcus elongatus PCC 7942

  • Compare growth rates and physiological responses under phosphate-replete and phosphate-limited conditions

  • Analyze phosphate uptake kinetics in wild-type versus mutant strains

  • Examine cellular phosphate content and polyphosphate accumulation

• Gene expression analysis:

  • Quantify Synpcc7942_1098 expression levels under varying phosphate concentrations using RT-qPCR or RNA-seq

  • Analyze co-expression patterns with known phosphate-responsive genes such as the pho regulon

  • Investigate potential regulatory relationships with the SphS/SphR two-component system, which is known to regulate phosphate response in cyanobacteria

• Protein interaction studies:

  • Perform co-immunoprecipitation or pull-down assays to identify potential interaction partners

  • Use bacterial two-hybrid systems to screen for interactions with components of phosphate sensing pathways

  • Employ crosslinking mass spectrometry to identify proximal proteins in vivo

• Physiological and biochemical characterization:

  • Measure alkaline phosphatase activity in wild-type versus mutant strains under phosphate limitation

  • Analyze membrane potential and ion flux in relation to phosphate transport

  • Investigate potential phosphorylation of Synpcc7942_1098 under different phosphate conditions

• Structural and localization studies:

  • Determine subcellular localization using fluorescently tagged Synpcc7942_1098

  • Investigate co-localization with known phosphate transporters or sensors

  • Analyze structural changes in response to phosphate availability using techniques like HDX-MS

The experimental design should include appropriate controls and consider the potential indirect effects of membrane protein manipulation on cellular physiology.

What are the major challenges in studying the function of uncharacterized membrane proteins like Synpcc7942_1098, and how can they be addressed?

Studying uncharacterized membrane proteins like Synpcc7942_1098 presents several unique challenges that require specialized approaches:

• Functional hypothesis generation:

  • Challenge: Without known homologs of characterized function, developing initial functional hypotheses is difficult.

  • Solution: Employ computational approaches like gene neighborhood analysis, co-expression network analysis, and structural prediction to generate testable hypotheses. Use comparative genomics to identify patterns in the distribution and conservation of the gene across different cyanobacterial species and environmental conditions.

• Phenotypic analysis of genetic mutants:

  • Challenge: Deletion mutants may not show clear phenotypes due to functional redundancy or complex regulatory networks.

  • Solution: Implement conditional mutants (inducible expression, repression) and double/triple mutants to overcome redundancy. Use high-throughput phenotyping approaches (e.g., Biolog plates) to test growth under hundreds of conditions simultaneously.

• Protein-protein interaction identification:

  • Challenge: Membrane protein interactions are difficult to detect using traditional methods like yeast two-hybrid.

  • Solution: Use membrane-specific interaction techniques such as membrane yeast two-hybrid (MYTH), proximity labeling methods (BioID, APEX), or split-ubiquitin systems. Validate interactions using in vivo approaches like FRET or BiFC.

• Developing functional assays:

  • Challenge: Without knowledge of function, designing specific activity assays is problematic.

  • Solution: Use untargeted approaches like metabolomics or lipidomics to detect changes associated with protein presence/absence. Monitor membrane properties (fluidity, potential) and transport activities across different conditions.

• Structural characterization:

  • Challenge: Membrane proteins are notoriously difficult to crystallize or study by traditional structural biology methods.

  • Solution: Employ advanced techniques like cryo-EM, solid-state NMR, or computer-aided structure prediction. Use hybrid approaches combining low-resolution experimental data with computational modeling.

A systematic workflow combining these approaches can substantially increase the chances of functional characterization success.

What system-wide approaches can be used to place Synpcc7942_1098 in the broader context of cyanobacterial cellular processes?

Employing system-wide approaches can provide valuable insights into the functional context of Synpcc7942_1098:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data from wild-type and Synpcc7942_1098 mutant strains

  • Analyze differential expression patterns across various environmental conditions (light, nutrient limitation, stress)

  • Construct gene regulatory networks to identify potential regulatory relationships

  • Apply machine learning approaches to identify patterns across multi-omics datasets

• Interactome mapping:

  • Perform systematic protein-protein interaction screens focusing on membrane protein complexes

  • Use complementary approaches like affinity purification-mass spectrometry (AP-MS) and proximity labeling

  • Develop an interaction map centered on Synpcc7942_1098 to identify functional modules

  • Validate key interactions using targeted approaches

• Comparative genomics across cyanobacterial species:

  • Analyze gene neighborhood conservation and co-evolution patterns

  • Identify correlated gene presence/absence across diverse cyanobacterial genomes

  • Examine evolutionary rate patterns to identify selection pressures

  • Correlate gene presence with specific ecological niches or metabolic capabilities

• Functional genomics screens:

  • Implement CRISPRi libraries to systematically identify genetic interactions

  • Perform chemical genomics to identify compounds with differential effects on wild-type versus mutant strains

  • Use transposon mutagenesis coupled with next-generation sequencing (Tn-Seq) to identify synthetic lethal or beneficial interactions

• Cellular physiology mapping:

  • Monitor changes in membrane potential, pH homeostasis, and ion fluxes

  • Assess effects on photosynthetic efficiency and electron transport

  • Examine changes in cell morphology and ultrastructure using advanced microscopy

The integration of these approaches through computational modeling can help place Synpcc7942_1098 in a broader functional context and generate testable hypotheses about its role in cyanobacterial physiology.

How can researchers optimize experimental conditions for functional characterization of Synpcc7942_1098?

Optimizing experimental conditions is crucial for successful functional characterization of membrane proteins like Synpcc7942_1098:

• Growth condition optimization:

  • Systematically vary key parameters including light intensity, spectral quality, temperature, CO2 concentration, and nutrient availability

  • Implement continuous culture systems (chemostats, turbidostats) to maintain precisely controlled steady-state conditions

  • Test responses to environmental stressors (oxidative stress, osmotic stress, metal toxicity)

  • Consider circadian effects by sampling across the diurnal cycle

• Protein expression and purification optimization:

  • Screen multiple detergents and lipid compositions for optimal protein stability

  • Test various buffer conditions using differential scanning fluorimetry to identify stabilizing factors

  • Optimize protein-to-lipid ratios for reconstitution experiments

  • Consider native nanodiscs or styrene maleic acid copolymer lipid particles (SMALPs) for extraction in native lipid environments

• Reconstitution system development:

  • Test different reconstitution methodologies (direct incorporation, detergent dialysis, rapid dilution)

  • Optimize lipid composition to mimic native cyanobacterial membranes

  • Consider directional reconstitution to maintain proper protein orientation

  • Verify functional reconstitution using activity assays or structural integrity measurements

• Assay condition optimization:

  • Develop high-throughput screening approaches to test multiple buffer conditions

  • Systematically vary pH, ionic strength, and specific ion concentrations

  • Screen potential substrates, cofactors, or regulatory molecules

  • Consider time-resolved measurements to capture transient activities or conformational changes

• Experimental design considerations:

  • Implement appropriate controls including inactive mutants (e.g., point mutations in predicted functional residues)

  • Use complementation experiments to verify phenotypic findings

  • Develop quantitative assays with adequate sensitivity and dynamic range

  • Consider combinatorial approaches testing multiple variables simultaneously

What emerging technologies could advance our understanding of Synpcc7942_1098 function and regulation?

Emerging technologies offer new opportunities to elucidate the function and regulation of uncharacterized membrane proteins like Synpcc7942_1098:

• Advanced cryo-EM methodologies:

  • Advances in sample preparation, detectors, and image processing now enable high-resolution structures of smaller membrane proteins

  • Time-resolved cryo-EM can capture different conformational states

  • In situ cryo-electron tomography can visualize proteins in their native membrane environment

• Single-molecule techniques:

  • Single-molecule FRET to study conformational dynamics

  • Patch-clamp fluorometry to correlate structural changes with function

  • Single-particle tracking to monitor diffusion and localization in native membranes

  • Optical tweezers or magnetic tweezers to study mechanical properties and interactions

• Advanced genetic manipulation tools:

  • CRISPR interference (CRISPRi) for tunable gene repression

  • Base editing for precise genetic modifications without double-strand breaks

  • Optogenetic and chemogenetic tools for temporal control of protein activity

  • Synthetic genetic circuits for dynamic regulation studies

• Artificial intelligence and machine learning:

  • Deep learning for structure prediction (AlphaFold2, RoseTTAFold)

  • Machine learning for functional annotation based on sequence patterns

  • Network analysis tools to identify functional relationships from large datasets

  • Automated experimental design to optimize conditions for functional characterization

• Advanced imaging technologies:

  • Super-resolution microscopy (PALM, STORM, STED) for nanoscale localization

  • Expansion microscopy for improved spatial resolution in complex samples

  • Correlative light and electron microscopy (CLEM) to combine functional and structural imaging

  • Label-free imaging techniques to observe native proteins without modification

These emerging technologies, when integrated with established approaches, can provide unprecedented insights into the structure, function, and regulation of membrane proteins like Synpcc7942_1098.

How might knowledge of Synpcc7942_1098 contribute to our understanding of cyanobacterial adaptation to environmental stresses?

Understanding the role of Synpcc7942_1098 could significantly impact our knowledge of cyanobacterial stress adaptation:

• Nutrient limitation responses:

  • If involved in phosphate sensing or transport, Synpcc7942_1098 could be crucial for adaptation to phosphate-limited environments

  • Comparisons with the known phosphate regulon components (SphS/SphR) could reveal novel regulatory mechanisms

  • Potential involvement in other nutrient (nitrogen, sulfur, metal) adaptation pathways could be investigated

• Membrane remodeling during stress:

  • As a membrane protein, Synpcc7942_1098 might participate in stress-induced membrane reorganization

  • Possible roles in maintaining membrane integrity during temperature, osmotic, or oxidative stress

  • Potential involvement in thylakoid membrane dynamics during light stress or state transitions

• Signaling and environmental sensing:

  • UPF0754 proteins might function as sensors for specific environmental parameters

  • Potential involvement in stress signal transduction pathways

  • Comparative analysis across cyanobacterial species from different habitats could reveal adaptive specializations

• Metabolic adaptation mechanisms:

  • Possible roles in metabolite transport or compartmentalization during stress

  • Involvement in energy allocation and resource management under resource limitation

  • Potential functions in carbon concentration mechanisms or photorespiration

• Evolutionary adaptation patterns:

  • Comparative genomics across cyanobacteria from diverse environments could reveal adaptive signatures

  • Analysis of selection pressure on different protein domains could identify stress-responsive regions

  • Investigation of horizontal gene transfer patterns could provide insights into ecological specialization

Research in this area could not only advance our fundamental understanding of cyanobacterial biology but also inform applications in biotechnology and sustainable agriculture where stress tolerance is a key consideration.

What interdisciplinary approaches could best advance our understanding of the structure-function relationship of Synpcc7942_1098?

Advancing our understanding of Synpcc7942_1098 structure-function relationships requires integrative approaches that bridge multiple disciplines:

• Integrative structural biology:

  • Combine multiple structural determination techniques (X-ray crystallography, cryo-EM, NMR, HDX-MS)

  • Integrate computational modeling with experimental data

  • Use molecular dynamics simulations to study protein dynamics in membrane environments

  • Apply cross-linking mass spectrometry to identify spatial relationships between domains

• Systems biology and bioinformatics:

  • Develop gene regulatory network models incorporating Synpcc7942_1098

  • Use machine learning to identify functional patterns from multi-omics data

  • Apply network analysis to position the protein within cellular pathways

  • Implement Bayesian approaches to integrate diverse data types for functional prediction

• Synthetic biology and protein engineering:

  • Design chimeric proteins to test domain-specific functions

  • Engineer sensors based on Synpcc7942_1098 to monitor cellular responses

  • Apply directed evolution to explore functional potential

  • Develop minimal systems for reconstitution of functional networks

• Environmental microbiology and ecology:

  • Study Synpcc7942_1098 homologs in environmental cyanobacterial isolates

  • Analyze distribution and variation patterns across different ecological niches

  • Correlate genetic variations with habitat-specific adaptations

  • Examine expression patterns in natural cyanobacterial communities

• Biotechnology and applied science:

  • Explore potential applications in biosensing or bioremediation

  • Investigate contributions to photosynthetic efficiency or stress tolerance

  • Develop engineered strains with modified Synpcc7942_1098 for specific applications

  • Explore potential biotechnological applications based on discovered functions

A collaborative research program integrating these interdisciplinary approaches would provide comprehensive insights into the structure-function relationships of Synpcc7942_1098 and potentially reveal novel aspects of cyanobacterial biology with both fundamental and applied implications.