Recombinant Hordeum vulgare Cytochrome c oxidase subunit 5C (COX5C)

What is the primary role of COX5C in barley mitochondrial function?

Cytochrome c oxidase subunit 5C (COX5C) in Hordeum vulgare functions as one of the nuclear-coded polypeptide chains of cytochrome c oxidase, which serves as the terminal oxidase in mitochondrial electron transport . The protein plays a critical role in the respiratory chain, participating in the electron transfer mechanism that ultimately catalyzes the reduction of oxygen to water. In barley, as in other plants, COX5C contributes to the assembly and stability of the cytochrome c oxidase complex (Complex IV), which is essential for oxidative phosphorylation. This process is particularly important during seed germination when there is a transition from predominantly fermentative metabolism to aerobic respiration . The protein consists of approximately 63 amino acids and coordinates with other subunits to maintain the structural integrity of the complex.

How does barley COX5C differ structurally from homologous proteins in other cereal crops?

Barley COX5C shares significant structural homology with its counterparts in other cereal crops, particularly rice (Oryza sativa), but exhibits distinct species-specific characteristics. Comparative analysis reveals that while the core functional domains responsible for electron transport are conserved, barley COX5C shows approximately 12-15% sequence divergence from rice COX5C . This divergence primarily occurs in the N-terminal region, which may influence interactions with species-specific assembly factors and regulatory proteins.

The structural differences include:

These structural variations may contribute to the adaptation of the respiratory chain to the specific metabolic requirements and environmental conditions faced by barley compared to rice and other cereals.

What are the critical controlled variables when designing experiments with recombinant barley COX5C?

• Expression system parameters: The choice of expression host (bacterial, yeast, insect, or plant-based), vector design, and induction conditions must be standardized across experimental replicates. For barley COX5C, codon optimization for the selected expression system is particularly important due to the distinct codon usage patterns in plant nuclear genes.

• Purification conditions: Buffer composition (pH, ionic strength, detergent concentration for membrane protein solubilization), temperature, and chromatography parameters must be kept constant, as these factors directly affect protein folding and activity .

• Storage conditions: Stability of recombinant COX5C is affected by storage temperature, buffer composition, and freeze-thaw cycles, which must be controlled to prevent activity loss between assays.

• Assay-specific variables: For enzymatic activity measurements, substrate concentrations, reaction time, temperature, and the presence of potential inhibitors or activators must be standardized .

• Protein concentration determination method: The same quantification method should be used throughout the study to avoid systematic errors in concentration measurements.

As a minimum, three relevant controlled variables must be maintained for reliable results, though five or more are recommended for complex biochemical studies of membrane-associated proteins like COX5C .

How should experimental controls be designed for functional studies of recombinant barley COX5C?

For functional studies of recombinant Hordeum vulgare COX5C, a robust experimental control framework is essential to validate findings and account for potential artifacts . The experimental control design should include:

• Standard of Comparison (SOC): The wild-type, native COX5C purified from barley mitochondria serves as the primary standard of comparison. This SOC should be characterized under identical conditions as the recombinant protein to determine whether recombinant production affects functional properties .

• Negative controls:

  • Expression host cells containing the empty vector processed through the same purification protocol

  • Heat-denatured recombinant COX5C to establish baseline for activity assays

  • Reactions without substrate or cofactors to detect background activity

• Positive controls:

  • Commercial cytochrome c oxidase from related species with established activity parameters

  • Reconstituted complex IV containing the recombinant COX5C subunit alongside other purified components

• Processing controls:

  • Parallel processing of samples to minimize batch effects

  • Time-course stability assessments to account for potential degradation during experimental procedures

The rationale for the SOC selection should be clearly documented, with preference given to controls that most closely approximate physiological conditions or that represent a null hypothesis baseline . For integration studies examining COX5C within the complete cytochrome c oxidase complex, assembling the complex with and without the recombinant subunit provides critical comparative data.

What expression systems yield optimal functional recombinant barley COX5C?

The selection of an appropriate expression system is crucial for obtaining functionally active recombinant Hordeum vulgare COX5C. Comparative analysis of expression systems reveals significant differences in yield, folding, and post-translational modifications:

The optimal expression system depends on the specific research objectives. For structural studies requiring large quantities, E. coli with specialized folding strains (Origami, SHuffle) provides sufficient yield, though activity may be compromised. For functional studies, Pichia pastoris offers a balance between yield and activity. The addition of specific chaperones and folding enhancers can significantly improve the functional properties of the recombinant protein regardless of the chosen system.

When expressing membrane-associated proteins like COX5C, the incorporation of detergent-compatible purification tags (such as His10 rather than standard His6) and careful optimization of membrane-mimicking environments during purification substantially improves functional recovery.

What are the most effective purification strategies for maintaining structural integrity of recombinant barley COX5C?

Purification of functionally active recombinant Hordeum vulgare COX5C requires careful consideration of its membrane-associated nature and sensitivity to denaturation. The most effective purification strategies incorporate:

• Initial extraction optimization: Gentle cell lysis using enzymatic methods (for yeast/insect cells) or specialized detergents like n-dodecyl β-D-maltoside (DDM) at 1-1.5% concentration preserves structural integrity better than mechanical disruption or harsh detergents like SDS .

• Multi-stage chromatography approach:

  • Initial capture using immobilized metal affinity chromatography (IMAC) with either Ni-NTA or TALON resins when using His-tagged constructs

  • Intermediate purification via ion exchange chromatography (typically anion exchange at pH 8.0)

  • Polishing step using size exclusion chromatography to remove aggregates and ensure conformational homogeneity

• Detergent exchange protocol: During purification, transitioning from extraction detergents to milder alternatives (like 0.1% DDM or 0.05% digitonin) maintains the native-like lipid environment critical for COX5C function.

• Stability enhancement additives:

  • Glycerol (10-15%) to prevent aggregation

  • Reducing agents (1-2 mM DTT or TCEP) to maintain thiol groups

  • Specific phospholipids (0.01-0.05 mg/ml cardiolipin) to stabilize protein-lipid interactions

• Quality control checkpoints: Regular monitoring of protein integrity using analytical size exclusion chromatography, circular dichroism spectroscopy, and thermal shift assays throughout the purification process identifies potential degradation early and allows for protocol adjustments.

This strategic approach typically yields 70-80% retention of functional activity compared to only 30-40% when using standard protocols designed for soluble proteins . The most critical step is maintaining an appropriate membrane-mimicking environment throughout purification to preserve the native structure of this mitochondrial protein.

How can protein-protein interaction networks of barley COX5C be effectively mapped?

Mapping the protein-protein interaction (PPI) network of Hordeum vulgare COX5C requires a multi-method approach to capture both stable and transient interactions within the mitochondrial respiratory complex. The most effective techniques include:

• Proximity-based labeling methods: BioID or APEX2 fused to recombinant COX5C enables in vivo labeling of proximal proteins through biotinylation, followed by streptavidin pulldown and mass spectrometry identification. This approach has successfully identified previously unknown interaction partners of COX5C, including assembly factors not detected by conventional methods .

• Cross-linking mass spectrometry (XL-MS): Chemical cross-linking using membrane-permeable reagents like DSS (disuccinimidyl suberate) followed by enzymatic digestion and specialized mass spectrometry analysis reveals direct contact points between COX5C and other subunits of the cytochrome c oxidase complex. This technique has identified specific residues involved in the interaction with COX1, the catalytic subunit of the enzyme complex .

• Co-immunoprecipitation with quantitative analysis: Antibody-based pulldown of COX5C followed by label-free quantitative proteomics has revealed differences in the interaction network during various developmental stages and stress conditions in barley . This approach is particularly valuable for detecting dynamic changes in the interactome.

• Yeast two-hybrid membrane system (MYTH): This specialized version of Y2H designed for membrane proteins has successfully mapped binary interactions between COX5C and both core complex components and peripheral regulatory factors.

The integration of data from these complementary approaches has revealed that barley COX5C interacts directly with:

• Core components: COX1, COX2, COX3, and COX4

• Assembly factors: COX11, COX17, and barley-specific chaperones

• Regulatory proteins: including stress-responsive factors unique to cereal crops

These interactions provide important insights into the role of COX5C beyond its structural contribution to the complex, suggesting regulatory functions in response to developmental cues and environmental conditions.

What spectroscopic techniques are most informative for structural characterization of recombinant barley COX5C?

Structural characterization of recombinant Hordeum vulgare COX5C requires specialized spectroscopic techniques that can accommodate its membrane-associated nature and complex interactions within the cytochrome c oxidase system:

• Circular Dichroism (CD) Spectroscopy: Far-UV CD (190-250 nm) provides essential information about the secondary structure composition, revealing that barley COX5C contains approximately 35% α-helical content and 15% β-sheet structures. Near-UV CD (250-350 nm) offers insights into the tertiary structure environment of aromatic residues and disulfide bonds. Temperature-dependent CD measurements indicate that recombinant barley COX5C exhibits a melting temperature (Tm) of 48-52°C, which is 3-5°C lower than the native protein isolated from barley mitochondria.

• Fourier Transform Infrared (FTIR) Spectroscopy: FTIR with attenuated total reflection (ATR) provides valuable complementary data on secondary structure in membrane environments. Analysis of the amide I band (1600-1700 cm⁻¹) confirms the mixed α-helical/β-sheet composition and reveals important information about protein-lipid interactions that are not accessible by other techniques.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: While challenging due to size constraints, selective isotopic labeling of recombinant COX5C (¹⁵N/¹³C) enables targeted NMR studies of specific regions, particularly the soluble domains. 2D heteronuclear single quantum coherence (HSQC) experiments have successfully mapped the conformational changes induced by interactions with other subunits of the complex.

• Electron Paramagnetic Resonance (EPR) Spectroscopy: Site-directed spin labeling of cysteine residues in recombinant COX5C provides critical information about local dynamics and accessibility. Double electron-electron resonance (DEER) measurements between strategically placed spin labels have been used to determine distance constraints within the protein structure.

• Resonance Raman Spectroscopy: This technique is particularly valuable for studying the interaction of COX5C with heme groups in the assembled complex, providing vibration frequencies sensitive to the redox state and coordination environment of the metal centers.

Each spectroscopic method provides unique and complementary structural information, with the integration of multiple techniques delivering the most comprehensive characterization. Of particular importance are detergent-screening experiments using these methods to identify conditions that maintain native-like structure, as inappropriate detergent selection can lead to structural perturbations that compromise functional interpretation.

How can aggregation issues during recombinant barley COX5C expression be overcome?

Aggregation of recombinant Hordeum vulgare COX5C presents a significant challenge during expression and purification, often resulting in inclusion body formation or non-functional protein. Systematic approaches to overcome these issues include:

• Expression parameter modification:

  • Reducing induction temperature to 16-18°C significantly decreases aggregation in E. coli systems by slowing protein synthesis and allowing more time for proper folding

  • Decreasing inducer concentration (e.g., 0.1 mM IPTG instead of standard 1 mM) to moderate expression rate

  • Using auto-induction media formulations to achieve gradual protein expression

• Co-expression strategies:

  • Integration of molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) increases soluble protein yield by 2.5-3 fold in bacterial systems

  • Co-expression with other cytochrome c oxidase subunits, particularly COX1 and COX4, provides stabilizing interactions that prevent COX5C aggregation

  • Addition of specific barley mitochondrial chaperones improves folding in heterologous systems

• Fusion tag optimization:

  • N-terminal fusion with MBP (maltose-binding protein) improves solubility more effectively than GST or SUMO tags for COX5C

  • Incorporation of a rigid linker (PAPAP sequence) between the tag and COX5C prevents interference with folding

  • Using dual tags (e.g., MBP at N-terminus and a small His-tag at C-terminus) facilitates both solubility and purification

• Buffer composition refinement:

  • Addition of non-detergent sulfobetaines (NDSB-201 at 0.5-1 M) reduces aggregation during cell lysis

  • Incorporation of specific lipids (0.1 mg/ml bovine heart cardiolipin) stabilizes the membrane-associated regions

  • Using arginine (50-100 mM) as a suppressor of protein-protein interactions during purification

• Refolding protocols for inclusion bodies:

  • High-pressure refolding (2000 bar) in the presence of detergent micelles achieves 40-50% recovery of functional protein

  • On-column refolding using immobilized metal affinity chromatography with a decreasing urea gradient (8M to 0M) while maintaining critical detergent concentrations

Implementation of these strategies has successfully increased the yield of correctly folded, functional recombinant barley COX5C from typical levels of 10-15% to 50-65% of total expressed protein . The most effective approach often combines multiple strategies, particularly temperature reduction, chaperone co-expression, and lipid supplementation.

What analytical methods best verify the functional integrity of purified recombinant barley COX5C?

Verification of functional integrity for purified recombinant Hordeum vulgare COX5C requires specialized analytical methods that assess both structural integrity and biological activity:

• Enzyme activity assays:

  • Cytochrome c oxidation kinetics measured spectrophotometrically at 550 nm provides direct assessment of electron transfer capacity

  • Oxygen consumption rates determined using Clark-type electrodes or optical sensors quantify the terminal oxidase function

  • Proton pumping efficiency measured in reconstituted proteoliposomes using pH-sensitive fluorescent dyes evaluates coupling efficiency

• Spectroscopic fingerprinting:

  • Absorption spectroscopy to verify characteristic Soret and α/β bands of properly incorporated heme groups

  • Resonance Raman spectroscopy targeting metal-ligand vibrational modes unique to functionally assembled cytochrome c oxidase

  • Electron paramagnetic resonance (EPR) to assess the redox centers in their native configuration

• Thermal and chemical stability profiles:

  • Differential scanning calorimetry (DSC) to determine melting temperatures of correctly folded protein

  • Thermal shift assays using environmentally sensitive fluorescent dyes for high-throughput stability screening

  • Chemical denaturation curves with detergents or chaotropic agents to quantify conformational stability

• Interaction verification:

  • Microscale thermophoresis (MST) to measure binding affinities with known interaction partners (COX1, COX4)

  • Surface plasmon resonance (SPR) to determine kinetic parameters of these interactions

  • Native mass spectrometry to confirm assembly into higher-order complexes

• Structural integrity assessment:

  • Limited proteolysis patterns compared to native protein isolated from barley mitochondria

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe solvent accessibility of different regions

  • Negative stain electron microscopy to visualize proper integration into the cytochrome c oxidase complex

Researchers should employ a combination of these methods, as no single technique provides comprehensive validation. The gold standard approach involves comparison of at least three orthogonal measures (typically one each from activity, spectroscopic, and structural categories) between the recombinant protein and native COX5C isolated from barley mitochondria . A functional recombinant protein should exhibit at least 75-80% of the activity of the native protein while maintaining similar spectroscopic signatures and thermal stability profiles.

How does COX5C gene expression vary during barley development and stress responses?

The expression pattern of Hordeum vulgare COX5C exhibits significant developmental regulation and environmental responsiveness, reflecting its critical role in mitochondrial function:

• Developmental expression profile:

  • Seed germination represents a key transition point, with COX5C expression increasing dramatically (8-12 fold) during the first 48 hours of imbibition as the seed transitions from fermentative to oxidative metabolism

  • Vegetative tissues show differential expression, with highest levels in metabolically active tissues (root tips, developing leaves) and lower levels in mature tissues

  • Reproductive development triggers tissue-specific expression patterns, with particularly high levels in developing anthers (20-fold higher than vegetative tissues) and moderate increases in developing embryos

• Diurnal and circadian regulation:

  • COX5C expression follows a distinct diurnal pattern with peak expression occurring 2-3 hours after dawn

  • This regulation persists under constant light conditions, confirming true circadian control rather than merely light-responsive expression

  • The amplitude of this oscillation varies by tissue type, with leaves showing the most pronounced rhythmicity

• Abiotic stress responses:

  • Cold stress (4°C) induces a biphasic response: an initial decrease (30-40%) during the first 6 hours followed by a compensatory increase (50-70% above baseline) after 24-48 hours

  • Drought stress causes tissue-specific regulation with upregulation in roots (2-3 fold) but minimal changes in leaf tissue

  • Salt stress (150 mM NaCl) triggers a rapid but transient increase (3-4 fold) in COX5C expression within 12 hours, returning to baseline after 48 hours

• Biotic stress responses:

  • Pathogen challenge (powdery mildew) induces moderate upregulation (1.5-2 fold) of COX5C in resistant varieties but minimal change in susceptible varieties

  • This differential response suggests a potential role in powering defense responses through enhanced mitochondrial capacity

• Hormonal regulation:

  • Abscisic acid treatment downregulates COX5C expression (40-50% reduction)

  • Gibberellic acid and auxin induce modest increases (20-30%)

  • Methyl jasmonate causes rapid but transient upregulation, potentially linking respiratory function to defense signaling

These expression patterns reveal COX5C as an environmentally responsive gene whose regulation is intricately linked to the metabolic demands of different developmental stages and stress responses in barley. The particularly strong induction during seed germination correlates with the critical role of mitochondrial biogenesis during this energy-demanding transition .

What genomic approaches are most effective for studying COX5C variants across barley cultivars?

Investigating COX5C variants across Hordeum vulgare cultivars requires specialized genomic approaches that account for the challenging nature of mitochondrial-nuclear gene interactions:

• Targeted sequencing strategies:

  • Long-read sequencing (PacBio or Oxford Nanopore) of the complete COX5C locus including 2 kb upstream and downstream regions provides comprehensive coverage of regulatory elements

  • Amplicon-based deep sequencing using multiplexed samples can efficiently screen large germplasm collections for common variants

  • RNA sequencing coupled with specific enrichment for low-abundance transcripts reveals splice variants and expression differences between cultivars

• Barley genomic resources integration:

  • The Barleymap platform enables precise positioning of COX5C variants on the current reference genomes (MorexV3)

  • Integration with POPSEQ and Barley Physical Map data provides alternative mapping frameworks for cultivars that diverge significantly from Morex

  • These complementary resources allow comprehensive assessment of syntenic conservation around the COX5C locus

• Variant classification and functional prediction:

  • Specialized algorithms for predicting the impact of non-synonymous substitutions on protein function must consider the unique constraints of mitochondrial proteins

  • Variants in barley COX5C can be classified into four categories based on their predicted impact:

    • Core functional domain mutations (typically deleterious)

    • Interface residue variations (potentially affecting complex assembly)

    • Surface variations (generally neutral)

    • Regulatory region polymorphisms (affecting expression rather than function)

• Population genomics approaches:

  • Genome-wide association studies (GWAS) linking COX5C haplotypes to phenotypic traits like growth rate, stress tolerance, and yield components

  • Selective sweep analysis identifies whether COX5C has been under selection during barley domestication or improvement

  • Haplotype network analysis reveals the evolutionary relationships between COX5C variants and their distribution across geographical regions

• Comparative genomics with wild relatives:

  • Sequencing COX5C from wild Hordeum species provides crucial evolutionary context for variant interpretation

  • Analysis of synonymous to non-synonymous substitution ratios reveals selective pressures on different regions of the gene

  • Identification of conserved non-coding sequences through multi-species alignments highlights critical regulatory elements

The most effective approach combines targeted long-read sequencing with comparative analysis across cultivated and wild barley accessions, integrated with the Barleymap platform for precise genomic positioning . This multi-faceted strategy has revealed surprising diversity in both coding and regulatory regions of COX5C across barley germplasm, with distinct haplotype groups associated with adaptation to different environmental conditions.

How can recombinant barley COX5C be utilized to study mitochondrial dysfunction phenotypes?

Recombinant Hordeum vulgare COX5C serves as a powerful tool for investigating mitochondrial dysfunction in cereals through several sophisticated research applications:

• In vitro reconstitution systems:

  • Purified recombinant COX5C can be incorporated into liposomes alongside other cytochrome c oxidase subunits to create functional minimal systems

  • These reconstituted complexes allow precise manipulation of subunit composition, enabling the introduction of specific mutations to study their functional consequences

  • Such systems have successfully recapitulated electron transfer and proton pumping activities, providing a platform to assess how variants affect core cytochrome c oxidase functions

• Complementation studies in mutant systems:

  • Barley lines with reduced COX5C expression show characteristic mitochondrial dysfunction phenotypes including reduced root growth, altered seed germination kinetics, and compromised stress responses

  • These phenotypes can be rescued through targeted expression of recombinant COX5C variants, allowing structure-function relationships to be tested in vivo

  • Complementation efficiency serves as a quantitative measure of functional impact for specific variants

• Protein-protein interaction network perturbation analysis:

  • Modified recombinant COX5C proteins with altered interaction surfaces can be used to systematically disrupt specific protein-protein interactions within the respiratory complex

  • This approach has identified previously unknown regulatory interactions between COX5C and stress-responsive factors that modulate respiratory efficiency under adverse conditions

  • Proximity labeling using recombinant COX5C fused to promiscuous biotin ligases (BioID2) reveals condition-specific changes in the interaction network

• Development of specific inhibitors and modulators:

  • Structure-based design of peptides that mimic COX5C interaction surfaces can selectively interfere with complex assembly

  • These tools enable precise temporal control of cytochrome c oxidase function to study the consequences of acute respiratory inhibition

  • The comparison between chronic (genetic) and acute (inhibitor-based) disruption provides insights into compensatory responses

• Biomarker development for mitochondrial dysfunction:

  • Antibodies raised against recombinant barley COX5C enable sensitive immunodetection of native protein

  • Changes in COX5C abundance, post-translational modifications, or subcellular distribution serve as early indicators of mitochondrial stress

  • These biomarkers have successfully identified mitochondrial dysfunction phenotypes before visible symptoms appear in plants exposed to specific agricultural chemicals

The integration of these approaches has revealed that even subtle alterations in COX5C structure or abundance can have profound effects on respiratory efficiency and stress responses in barley. Particularly significant is the discovery that certain COX5C variants confer enhanced tolerance to specific stresses through altered interaction with regulatory factors, suggesting potential targets for crop improvement efforts .

What computational approaches best predict the impact of mutations in barley COX5C?

Computational prediction of mutation impacts in Hordeum vulgare COX5C requires specialized approaches that account for both its role in protein complexes and the evolutionary constraints on mitochondrial proteins:

The most effective prediction pipeline combines evolutionary conservation analysis, all-atom molecular dynamics simulations, and machine learning integration, achieving a correlation of r=0.74 between predicted and experimentally measured functional impacts for a validation set of 28 COX5C variants. This integrated approach has successfully identified subtle mutation effects that were missed by traditional sequence-based methods alone.

How does post-translational modification affect barley COX5C function?

Post-translational modifications (PTMs) of Hordeum vulgare COX5C play critical yet understudied roles in regulating respiratory chain function and mitochondrial adaptation to changing conditions:

• Phosphorylation landscape:

  • Mass spectrometry analysis has identified four phosphorylation sites in barley COX5C (Ser-11, Thr-15, Ser-36, and Thr-51)

  • Phosphorylation at Ser-36 increases dramatically (4-6 fold) during seed germination, correlating with the activation of respiratory metabolism

  • Site-directed mutagenesis of these residues to phosphomimetic (Asp/Glu) or non-phosphorylatable (Ala) variants has revealed that:

    • Ser-36 phosphorylation enhances interaction with COX4, promoting complex assembly

    • Thr-51 phosphorylation reduces catalytic efficiency by approximately 25-30%

    • The phosphorylation state of Ser-11 alters the protein's half-life in vivo

• Acetylation regulation:

  • Lysine acetylation at positions K40 and K47 has been detected in barley COX5C

  • The acetylation pattern shows circadian rhythmicity, with peak acetylation occurring during the night phase

  • This modification appears to be responsive to cellular carbon status, potentially linking respiratory efficiency to carbon availability

• Redox-sensitive modifications:

  • The two conserved cysteine residues in barley COX5C (Cys-24 and Cys-68) can form a reversible disulfide bond under oxidizing conditions

  • This redox switch alters the conformation of COX5C, reducing its affinity for other complex subunits by approximately 60%

  • The redox state of these cysteines thus serves as a molecular sensor linking environmental oxidative stress to respiratory chain modulation

• Developmental and stress-responsive PTM patterns:

  • The PTM profile of COX5C changes significantly during the transition from heterotrophic to autotrophic growth in seedlings

  • Drought stress induces a distinct pattern of modifications, particularly increased phosphorylation at Thr-15

  • Cold stress triggers accumulation of the acetylated form, potentially as a mechanism to fine-tune respiratory efficiency at low temperatures

• PTM crosstalk mechanisms:

  • Phosphorylation at Ser-11 enhances subsequent acetylation at K40, revealing hierarchical modification patterns

  • The redox state of the Cys-24/Cys-68 pair influences accessibility of Thr-51 to kinases, creating a complex regulatory network

  • This crosstalk creates condition-specific PTM signatures that precisely calibrate COX5C function