Recombinant Oenothera parviflora Chloroplast envelope membrane protein (cemA)

Structural and Functional Characteristics

Q: What is the cemA protein and what is its biological function in Oenothera species?

A: The cemA protein (Chloroplast envelope membrane protein) is a 214-amino acid membrane-bound protein localized to the chloroplast envelope in Oenothera species. It plays a critical role in chloroplast function and is encoded by the chloroplast genome. The protein is characterized by its hydrophobic domains that facilitate membrane integration. While its complete functional profile remains under investigation, current evidence suggests involvement in CO₂ uptake processes and potential roles in chloroplast membrane integrity . The protein belongs to the broader Onagraceae family, which includes evening primroses that have been extensively studied for their unique genetic and evolutionary characteristics .

Q: What is the amino acid sequence and predicted structure of the Oenothera parviflora cemA protein?

A: The Oenothera parviflora cemA protein consists of 214 amino acids with the following sequence:
MVFFPWWISLLFNKGLESWVTNWWNTTHSETFLTDMQEKSILDKFIELEELLLLDEMINE
YPETHLQTLRIGIHKEMVRLIKMRNEDHIHTILHLSTNIICFIIFRGYSILGNKELLILN
SWMQEFLYNLSDTIKAFSILLLTDFCIGFHSPHGWELMIAYVYKDFGFAQNDQIISGLVS
TFPVILDTIFKYWIFRYLNRVSPSLVVIYDSMND

Structural analysis reveals multiple hydrophobic regions consistent with its membrane-spanning function. The protein contains characteristic motifs common to chloroplast membrane proteins, including transmembrane helices that anchor it within the chloroplast envelope. Secondary structure prediction algorithms suggest approximately 40-45% alpha-helical content with limited beta-sheet structures, typical of membrane-associated proteins. The protein has a UniProt accession number of B0Z5E0 .

Production and Storage Parameters

Q: What expression systems are most effective for recombinant cemA protein production?

A: Recombinant cemA protein is most effectively produced in E. coli expression systems. For optimal expression, the full-length sequence (amino acids 1-214) is typically used with an N-terminal His-tag to facilitate purification . Expression optimization requires careful consideration of induction parameters, with IPTG concentration typically maintained between 0.5-1.0 mM and induction temperatures of 18-25°C to minimize inclusion body formation. Alternative expression systems such as insect cells may be employed for studies requiring post-translational modifications, though bacterial systems remain the standard for basic structural and functional studies. The expression region encompasses the entire coding sequence from positions 1-214 to ensure complete protein functionality .

Q: What are the optimal storage conditions for maintaining cemA protein stability?

A: For optimal stability, recombinant cemA protein should be stored at -20°C for short-term use and -80°C for extended storage . The protein is typically provided in a Tris-based buffer containing 50% glycerol, which has been optimized to maintain structural integrity during freeze-thaw cycles . Working aliquots may be stored at 4°C for up to one week to minimize degradation from repeated freeze-thaw cycles . When handling the protein, it is recommended to briefly centrifuge vials before opening to ensure contents are collected at the bottom. For lyophilized preparations, reconstitution should be performed using deionized sterile water to a concentration of 0.1-1.0 mg/mL, followed by addition of glycerol to a final concentration of 30-50% for cryoprotection during storage .

Experimental Design Considerations

Q: How can researchers effectively design experiments to investigate cemA protein interactions with other chloroplast components?

A: Designing experiments to investigate cemA protein interactions requires a multi-methodological approach. Begin with in vitro pull-down assays using the recombinant His-tagged cemA protein as bait to identify potential interaction partners from chloroplast extracts. Follow with co-immunoprecipitation studies using antibodies specific to the cemA protein or its tag. For higher-resolution analysis, implement techniques such as:

• Bimolecular Fluorescence Complementation (BiFC) to visualize interactions in planta

• Surface Plasmon Resonance (SPR) to determine binding kinetics

• Isothermal Titration Calorimetry (ITC) for thermodynamic profiling of interactions

• Crosslinking Mass Spectrometry (XL-MS) to identify interaction interfaces

Include appropriate controls in each experiment, such as non-interacting chloroplast proteins and mutated versions of cemA lacking key domains. When reconstituting the protein for interaction studies, ensure it maintains its native conformation by verifying proper folding through circular dichroism spectroscopy . The amino acid sequence suggests several potential protein-protein interaction motifs that can guide targeted mutation studies to confirm specificity of observed interactions.

Q: What approaches are recommended for studying the role of cemA in comparative analyses across Oenothera species?

A: For comparative analyses of cemA across Oenothera species, researchers should implement a systematic approach combining genomic, biochemical, and functional methodologies:

• Sequence Analysis Pipeline:

  • Perform multiple sequence alignments of cemA sequences from various Oenothera species, including O. parviflora and O. argillicola

  • Calculate conservation scores for each amino acid position

  • Identify species-specific substitutions that may correlate with phenotypic differences

  • Construct phylogenetic trees to visualize evolutionary relationships

• Structural Comparison:

  • Express and purify cemA proteins from different species

  • Compare their biophysical properties using techniques such as circular dichroism and thermal shift assays

  • Map sequence differences onto predicted structural models

• Functional Assessment:

  • Develop complementation assays using cemA mutants

  • Quantify photosynthetic efficiency parameters across species

  • Measure CO₂ uptake capabilities in controlled experimental systems

The Oenothera genus is particularly suited for such comparative studies due to its genetically distinct plastome types and unique evolutionary history . The data from such studies can be integrated with information on plant adaptation to different environments, as Oenothera species show substantial ecological diversity while maintaining high conservation in chloroplast proteins .

Technical Challenges and Solutions

Q: What strategies can address the challenges in maintaining cemA protein solubility during experimental procedures?

A: Maintaining cemA protein solubility presents significant challenges due to its hydrophobic nature as a membrane protein. Implement these research-validated strategies:

• Buffer Optimization:

  • Use buffers containing mild detergents (0.1-1% n-Dodecyl β-D-maltoside or CHAPS)

  • Include glycerol (10-30%) to prevent aggregation

  • Maintain pH between 7.0-8.0 to optimize solubility

  • Consider adding stabilizing agents such as arginine (50-100 mM)

• Temperature Management:

  • Perform all handling procedures at 4°C

  • Avoid rapid temperature fluctuations

  • For longer experiments, use cooling systems to maintain low temperatures

• Concentration Considerations:

  • Keep protein concentrations below 1 mg/mL during experimental procedures

  • If higher concentrations are required, add solubilizing agents incrementally

  • Use centrifugation (14,000 × g for 10 minutes) before experiments to remove any insoluble aggregates

• Reconstitution Approaches:

  • For functional studies, consider reconstitution into liposomes or nanodiscs

  • Use gradual dialysis to remove detergents when transitioning buffers

  • Validate membrane insertion using protease protection assays

When working with reconstituted protein, validate proper folding using intrinsic tryptophan fluorescence spectroscopy, as the cemA sequence contains multiple tryptophan residues that can serve as structural probes .

Q: How can researchers effectively troubleshoot expression and purification issues with recombinant cemA protein?

A: Troubleshooting expression and purification of recombinant cemA requires systematic problem identification and targeted solutions:

When expressing the full-length protein (1-214), monitor growth curves closely, as overexpression can lead to growth arrest. Consider using fusion partners like MBP or SUMO to enhance solubility, with subsequent tag removal using specific proteases . For challenging preparations, membrane scaffold proteins can be co-expressed to stabilize the membrane domains of cemA.

Protein Characterization Techniques

Q: What spectroscopic methods are most informative for characterizing the structural properties of recombinant cemA protein?

A: For comprehensive structural characterization of recombinant cemA protein, researchers should employ a combination of complementary spectroscopic techniques:

• Circular Dichroism (CD) Spectroscopy:

  • Far-UV CD (190-250 nm) to quantify secondary structure elements

  • Near-UV CD (250-350 nm) to assess tertiary structure fingerprints

  • Thermal denaturation CD to determine structural stability (melting temperature)

• Fluorescence Spectroscopy:

  • Intrinsic tryptophan fluorescence to monitor folding status

  • Bis-ANS binding assays to evaluate exposed hydrophobic surfaces

  • FRET-based assays to measure intramolecular distances and conformational changes

• Fourier-Transform Infrared Spectroscopy (FTIR):

  • Particularly valuable for membrane proteins like cemA

  • Provides information on secondary structure in membrane environments

  • Can be performed in detergent micelles or reconstituted proteoliposomes

• Nuclear Magnetic Resonance (NMR):

  • For specific domain analysis if complete structure determination is challenging

  • 15N-HSQC experiments to assess protein folding and ligand binding

  • Solid-state NMR for membrane-embedded structural analysis

When interpreting spectroscopic data, consider the native membrane environment of cemA. The protein's sequence contains multiple aromatic amino acids that serve as excellent intrinsic probes for folding studies . For proteins expressed with His-tags, ensure that tag effects on structure are accounted for in analyses by comparing with tag-cleaved preparations when possible.

Functional Assays

Q: What assays can be employed to assess the functional integrity of recombinant cemA protein?

A: Assessing the functional integrity of recombinant cemA protein requires specialized assays that address its role in chloroplast membrane function:

• Reconstitution Activity Assays:

  • Incorporate purified cemA into liposomes containing appropriate lipid compositions

  • Measure ion conductance or small molecule transport across these proteoliposomes

  • Compare activity with native chloroplast envelope preparations

• CO₂ Uptake Measurements:

  • Based on cemA's proposed role in carbon uptake

  • Use radioisotope-labeled carbon dioxide (14CO₂) to track transport

  • Implement membrane vesicle transport assays with reconstituted protein

• Binding Partner Interaction Assays:

  • Develop pull-down assays with known or predicted interaction partners

  • Quantify binding affinities using microscale thermophoresis or SPR

  • Verify interactions using crosslinking followed by mass spectrometry

• Membrane Integration Verification:

  • Protease protection assays to confirm proper membrane topology

  • Sucrose gradient fractionation to verify association with membrane fractions

  • Fluorescence-based membrane insertion assays using environment-sensitive probes

For all functional assays, parallel experiments should be conducted with site-directed mutants affecting key functional residues to validate specificity. When designing these experiments, the highly conserved sequence between different Oenothera species (such as O. parviflora and O. argillicola) suggests functional importance of these conserved regions . The similarity in amino acid sequence between these species (MVFFPWWISLLFNKGLESWVTNWWNTTHSETFLTDMQEKSILDKFIELEELLLLDEMINE YPETHLQTLRIGIHKEMVRLIKMRNEDHIHTILHLSTNIICFIIFRGYSILGNKELLILN SWMQEFLYNLSDTIKAFSILLLTDFCIGFHSPHGWELMIAYVYKDFGFAQNDQIISGLVS TFPVILDTIFKYWIFRYLNRVSPSLVVIYDSMND) indicates conserved functional domains that should be prioritized in mutational analyses.

Comparative Genomics Applications

Q: How can cemA protein studies contribute to understanding chloroplast genome evolution across Onagraceae?

A: The cemA protein serves as an excellent molecular marker for investigating chloroplast genome evolution across the Onagraceae family due to several key characteristics:

• Phylogenetic Signal Analysis:

  • The cemA gene shows sufficient sequence variation between species to resolve evolutionary relationships

  • Comparison of synonymous versus non-synonymous substitution rates can reveal selection pressures

  • Alignment of cemA sequences from diverse Oenothera species can identify lineage-specific adaptations

• Chloroplast Genome Structure Studies:

  • Analyze the genomic context of cemA in different Oenothera species

  • Examine conservation of gene order and intergenic regions

  • Investigate potential gene transfer events between chloroplast and nuclear genomes

• Functional Evolution Framework:

  • Compare cemA protein function across species with different photosynthetic adaptations

  • Correlate sequence changes with ecological niches of different Oenothera species

  • Develop models explaining how membrane protein evolution contributes to chloroplast adaptability

The genus Oenothera is particularly valuable for such studies due to its unique genetic system and the presence of genetically distinct plastome types . The five genetically distinct plastome types found in Oenothera provide natural variation that can be exploited to understand how chloroplast membrane proteins evolve in response to different selective pressures. These comparative genomic approaches should incorporate data from both O. parviflora and related species like O. argillicola to maximize evolutionary insights .

Structure-Function Relationship Studies

Q: What mutagenesis strategies are most informative for investigating cemA protein structure-function relationships?

A: Systematic mutagenesis of cemA protein provides critical insights into structure-function relationships through these research-validated approaches:

• Alanine Scanning Mutagenesis:

  • Systematically replace clusters of 3-5 amino acids with alanine across the entire sequence

  • Focus particularly on the highly conserved regions between different Oenothera species

  • Assess functional consequences using reconstitution assays

  • Create an activity map correlating sequence positions with functional importance

• Conservation-Guided Targeted Mutagenesis:

  • Identify residues conserved across all Oenothera species but divergent from other genera

  • Prioritize charged and aromatic residues in predicted functional domains

  • Design reciprocal mutations between divergent species to test function swapping

• Domain Swap Experiments:

  • Exchange putative functional domains between cemA and related proteins

  • Create chimeric proteins with other chloroplast membrane proteins

  • Assess which domains confer species-specific functional properties

• Topology Manipulation:

  • Introduce or remove predicted membrane-spanning regions

  • Modify charged residues at membrane interfaces

  • Assess how changes affect membrane integration and protein function

Implementation of these strategies should include both in vitro and in vivo functional assays. For in vitro work, purify each mutant protein using identical protocols to ensure comparable quality. For in vivo studies, complement cemA-deficient plants to assess functional restoration. All experiments should include wild-type controls processed in parallel to normalize results . The amino acid sequence provided in search results (MVFFPWWISLLFNKGLESWVTNWWNTTHSETFLTDMQEKSILDKFIELEELLLLDEMINE YPETHLQTLRIGIHKEMVRLIKMRNEDHIHTILHLSTNIICFIIFRGYSILGNKELLILN SWMQEFLYNLSDTIKAFSILLLTDFCIGFHSPHGWELMIAYVYKDFGFAQNDQIISGLVS TFPVILDTIFKYWIFRYLNRVSPSLVVIYDSMND) contains multiple regions that should be prioritized for mutagenesis, particularly the highly conserved transmembrane domains.

Quality Control Procedures

Q: What quality control metrics should be implemented when working with recombinant cemA protein preparations?

A: Implementing rigorous quality control metrics for recombinant cemA protein preparations is essential for experimental reproducibility:

• Purity Assessment:

  • SDS-PAGE analysis with minimum purity threshold of 90%

  • Western blotting with antibodies against the protein and/or tag

  • Mass spectrometry to confirm identity and detect post-translational modifications

  • Size exclusion chromatography to evaluate oligomeric state and aggregation propensity

• Structural Integrity Verification:

  • Circular dichroism to confirm expected secondary structure content

  • Fluorescence spectroscopy to assess tertiary structure

  • Limited proteolysis to verify proper folding (properly folded proteins show distinct digestion patterns)

  • Thermal shift assays to determine stability profiles

• Functional Validation:

  • Binding assays with known interaction partners

  • Activity assays specific to cemA function

  • Comparison with native protein when available

  • Batch-to-batch consistency testing

• Storage Stability Monitoring:

  • Accelerated stability testing at elevated temperatures

  • Time-course analysis of activity and structural integrity

  • Freeze-thaw cycle tolerance assessment

  • Monitoring of solutions for visible precipitation or turbidity

Document all quality control data in laboratory records with specific acceptance criteria for each parameter. For recombinant preparations, verify the complete amino acid sequence periodically using mass spectrometry or N-terminal sequencing. Implement these procedures consistently between batches to ensure experimental reproducibility .

Experimental Design Controls

Q: What are the essential controls for experiments investigating cemA protein function in chloroplast membrane studies?

A: Rigorous experimental design for cemA functional studies requires comprehensive controls at multiple levels:

• Protein-Level Controls:

  • Inactive cemA mutants (site-directed mutations in predicted functional residues)

  • Heat-denatured cemA protein to control for non-specific effects

  • Related but functionally distinct chloroplast membrane proteins

  • Tag-only preparations to control for tag-mediated effects

• System-Level Controls:

  • Empty liposomes/membranes without reconstituted protein

  • Membranes with irrelevant control proteins of similar size/structure

  • Gradient of cemA concentrations to establish dose-dependent effects

  • Time-course measurements to distinguish kinetic differences

• Validation Controls:

  • Parallel experiments with native chloroplast preparations

  • Complementation assays in cemA-deficient systems

  • Inhibitor studies with compounds targeting similar membrane processes

  • Cross-species comparisons using cemA from multiple Oenothera species

• Technical Controls:

  • Buffer-only samples to establish baselines

  • Internal standards for quantitative measurements

  • Replicate samples to assess experimental variability

  • Independent methods to verify key findings

When designing experiments with recombinant cemA protein, always include both positive and negative controls processed identically to experimental samples. For membrane integration studies, carefully control detergent concentrations as they can significantly impact results. When comparing results between Oenothera species, account for potential differences in expression levels and post-translational modifications .