Recombinant Gloeobacter violaceus NAD (P)H-quinone oxidoreductase subunit J (ndhJ)

What makes Gloeobacter violaceus significant in evolutionary studies of photosynthesis?

Gloeobacter violaceus represents an evolutionary primordial cyanobacterium with unique ancestral cell organization. It is significant because it:

• Completely lacks inner membranes (thylakoids) while all other cyanobacteria and chloroplasts have them

• Has photosynthetic apparatus located in the plasma membrane rather than in specialized thylakoid membranes

• Occupies a basal position in phylogenetic analyses among all organisms capable of plant-like photosynthesis

• Shows unique energy transfer pathways in its light-harvesting systems

As noted by numerous phylogenetic studies, G. violaceus has become a key species in evolutionary research of photosynthetic life . Its primitive features provide insights into early photosynthetic mechanisms that evolved approximately 3.2-3.7 billion years ago, making proteins like ndhJ particularly valuable for understanding the evolution of electron transport chains .

What is the structural and functional characterization of ndhJ in Gloeobacter violaceus?

The ndhJ protein (UniProt No. Q7NML5) is a subunit of the NAD(P)H-quinone oxidoreductase complex with the following characteristics:

• Consists of 176 amino acids with the sequence: MEEQTTQSAADGQTAIELVTGPISDALKARGLPHELTGLDNRKIEIIKVEPEHLIAVARALYDDGFNYLACQCGFDEGPGDSLGSMYHLTKLSDSADRPPEVRIKVFLPRDNPRVPSVYWIWKTADWQERESFDMYGIIYEGHPNLIRILMPEDWVGWPMRKDYVTPDFYELQDAY

• Functions as part of the electron transport chain in the plasma membrane

• Contains regions involved in NADPH binding and quinone reduction

• Unlike typical cyanobacterial NDH complexes that function in thylakoids, the G. violaceus ndhJ operates in the plasma membrane

The protein has been classified under EC 1.6.5.- and is alternatively known as "NAD(P)H dehydrogenase subunit J" or "NADH-plastoquinone oxidoreductase subunit J" .

What are the optimal conditions for expression and purification of recombinant Gloeobacter violaceus ndhJ?

Based on established protocols for recombinant ndhJ production:

• Expression system: E. coli is the preferred heterologous expression system, similar to methods used for other G. violaceus proteins

• Purification approach:

  • His-tag affinity chromatography is most commonly used

  • Typical yield is >85% purity as determined by SDS-PAGE

• Buffer conditions:

  • Tris-based buffer with 50% glycerol for stability

  • pH 7.2-8.0 is optimal for maintaining protein integrity

• Storage recommendations:

  • Store at -20°C/-80°C for long-term storage (shelf life of liquid form: 6 months; lyophilized form: 12 months)

  • Working aliquots can be stored at 4°C for up to one week

  • Avoid repeated freeze-thaw cycles

For reconstitution, it's recommended to use deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol added as a cryoprotectant .

What techniques are most effective for assessing ndhJ activity in vitro?

Several complementary approaches are recommended for characterizing recombinant ndhJ activity:

• Spectrophotometric assays:

  • NADPH oxidation can be monitored by the decrease in absorbance at 340 nm

  • Quinone reduction can be monitored by absorbance changes specific to the quinone substrate

  • Rate calculations should account for the extinction coefficient of NADPH (6,220 M⁻¹cm⁻¹)

• Oxygen consumption measurements:

  • Using oxygen electrodes to measure rate of electron transport activity

  • Standard conditions: pH 7.5, 30°C, 100-200 μM NADPH, and appropriate quinone acceptors

• Electron paramagnetic resonance (EPR):

  • For detection of transient radical species during electron transfer

  • Important for elucidating electron transfer mechanism within the complex

When interpreting activity data, it's essential to consider that ndhJ functions as part of a multi-subunit complex, and isolation may impact native activity levels.

How does the ndhJ from Gloeobacter violaceus differ from homologous proteins in thylakoid-containing cyanobacteria?

Comparative analyses reveal several key differences:

These differences reflect adaptations to the unique membrane architecture of G. violaceus and provide insights into how NDH complexes evolved alongside thylakoid membrane development . The primitive features of G. violaceus ndhJ make it a valuable model for understanding the ancestral functions of these proteins before the evolution of specialized thylakoid membranes.

What are the implications of studying G. violaceus ndhJ for understanding the evolution of photosynthetic electron transport chains?

G. violaceus ndhJ research offers several significant insights:

• Evolutionary trajectory: As G. violaceus represents the earliest diverging lineage of extant cyanobacteria, its ndhJ provides a window into the ancestral state of electron transport components before thylakoid evolution .

• Adaptation to membrane environments: The protein reveals how electron transport complexes functioned in primitive plasma membrane-localized photosynthesis compared to thylakoid-based systems .

• Functional conservation: Despite structural differences, the fundamental electron transport function has been conserved, demonstrating the essential nature of these processes across evolutionary time .

• Unique energy coupling mechanisms: G. violaceus has a distinct architecture where H⁺ pumping by NDH complex occurs across the plasma membrane rather than thylakoid membrane, providing insights into how early photosynthetic organisms generated proton motive force .

Research shows that G. violaceus and other primitive cyanobacteria likely evolved in rock-associated, calcifying biofilm habitats such as stromatolites or travertine spring mats , suggesting environmental factors that may have shaped early electron transport systems.

How can site-directed mutagenesis of ndhJ contribute to understanding quinone binding and electron transfer mechanisms?

Site-directed mutagenesis studies of ndhJ can reveal critical insights about structure-function relationships:

• Key residues for investigation:

  • Conserved motifs in NADPH binding domain

  • Putative quinone interaction sites

  • Residues at interfaces with other NDH subunits

  • Areas showing evolutionary divergence from other cyanobacteria

• Experimental approaches:

  • Create systematic mutations of targeted residues

  • Express mutant proteins and assess changes in:

    • NADPH binding affinity (using isothermal titration calorimetry)

    • Electron transfer rates (using stopped-flow spectroscopy)

    • Complex assembly (using native PAGE and crosslinking)

    • Quinone reduction kinetics (using specialized electrochemical methods)

• Expected outcomes:

  • Identification of residues essential for catalytic function

  • Understanding of how primitive ndhJ coordinates electron transfer

  • Insights into evolutionary changes in electron transport mechanisms

This approach has been successful in characterizing other electron transport components in cyanobacteria, where specific amino acid substitutions revealed the molecular basis for functional differences between primitive and advanced photosynthetic systems .

How can researchers overcome solubility and stability issues when working with recombinant G. violaceus ndhJ?

Several strategies have proven effective:

• Solubility enhancement:

  • Expression as fusion proteins with solubility tags (MBP, SUMO, or thioredoxin)

  • Co-expression with chaperones (GroEL/GroES system)

  • Lowering expression temperature (16-18°C)

  • Using specialized E. coli strains optimized for membrane protein expression

• Stability optimization:

  • Inclusion of 50% glycerol in storage buffer

  • Addition of reducing agents (1-5 mM DTT or β-mercaptoethanol)

  • Use of mild detergents (0.02% n-dodecyl-β-D maltopyranoside) when working with membrane-associated forms

  • Avoidance of repeated freeze-thaw cycles

• Functional reconstitution:

  • Assembly with other NDH subunits to form functional subcomplexes

  • Incorporation into liposomes or nanodiscs to mimic native membrane environment

  • Addition of native lipids from G. violaceus to stabilize protein structure

These approaches have successfully addressed similar challenges in working with other membrane-associated proteins from G. violaceus, such as rhodopsins .

What are the challenges in reconstituting functional NAD(P)H-quinone oxidoreductase complexes containing ndhJ?

Reconstitution of functional complexes faces several challenges:

• Subunit stoichiometry:

  • Determining the correct ratio of ndhJ to other NDH subunits

  • Identifying all necessary components for minimal functional activity

• Membrane integration:

  • Proper insertion into artificial membranes or nanodiscs

  • Maintaining correct orientation of the complex

• Assessing activity:

  • Developing reliable assays for the reconstituted complex

  • Distinguishing between partial and complete electron transfer activities

• Reproducibility issues:

  • Batch-to-batch variation in complex formation

  • Maintaining stability of the assembled complex

Successful reconstitution strategies include step-wise assembly protocols, use of mild detergents during the reconstitution process, and validation of complex integrity through multiple complementary techniques such as blue native PAGE, size-exclusion chromatography, and electron microscopy.

How does research on G. violaceus ndhJ inform our understanding of chloroplast evolution and endosymbiosis?

Research on G. violaceus ndhJ provides valuable insights into chloroplast evolution:

• Pre-endosymbiotic state: G. violaceus represents the most primitive extant cyanobacteria, offering a glimpse into the likely characteristics of pre-endosymbiotic cyanobacteria that eventually gave rise to chloroplasts .

• Conservation of electron transport components: Comparing ndhJ between G. violaceus, other cyanobacteria, and chloroplasts reveals which features were retained and which were modified during endosymbiosis.

• Membrane adaptation: The transition from plasma membrane-localized to thylakoid-localized electron transport in G. violaceus versus other cyanobacteria parallels the evolutionary changes that occurred during chloroplast development .

• Gene transfer patterns: Analysis of ndhJ gene sequences helps track the evolutionary history of gene transfers from the endosymbiont to the host nucleus during chloroplast evolution.

According to evolutionary analyses, G. violaceus diverged before the ancestor of chloroplasts, making its ndhJ an important outgroup for understanding which features of electron transport complexes were present in the cyanobacterial ancestor of chloroplasts versus those that evolved later .

What insights can G. violaceus ndhJ research provide for synthetic biology applications in alternative electron transport chains?

G. violaceus ndhJ research offers several applications for synthetic biology:

• Minimalist electron transport systems: The primitive nature of G. violaceus electron transport provides a blueprint for designing simplified artificial electron transport chains with fewer components than modern cyanobacterial or plant systems.

• Novel membrane adaptations: Understanding how ndhJ functions in the plasma membrane could inform the design of artificial electron transport systems in non-thylakoid membranes or synthetic membranes.

• Robust electron transport modules: G. violaceus has evolved to function in extreme environments (limestone surfaces, high light, desiccation cycles) , suggesting its electron transport components may be more robust for certain synthetic biology applications.

• Alternative energy coupling mechanisms: The unique H+ pumping mechanism in G. violaceus provides inspiration for novel ways to generate proton gradients in synthetic systems.

• Engineering electron transfer specificity: Structural insights from ndhJ could guide protein engineering to create variants with altered quinone specificity or coupling efficiency.

These applications are particularly relevant for synthetic biology projects aimed at creating artificial photosynthetic systems or modifying electron transport for bioproduction of energy carriers and high-value compounds.