Recombinant Prochlorococcus marinus ATP synthase subunit beta (atpD)

What is the role of ATP synthase subunit beta (atpD) in Prochlorococcus marinus?

The ATP synthase subunit beta (atpD) in Prochlorococcus marinus is a crucial component of the F₀F₁ ATP synthase complex, responsible for ATP production during oxidative phosphorylation. As part of the F₁ catalytic domain, the beta subunit contains nucleotide-binding sites and participates directly in ATP synthesis. Recent research has shown that ATP synthase may have additional roles beyond energy production, including potential involvement in lipid metabolism and transfer of lipids to carrier proteins, similar to what has been observed in other organisms . In cyanobacteria, the ATP synthase complex is particularly important for balancing energy needs during light and dark cycles, with specific inhibitory mechanisms to prevent ATP hydrolysis under certain conditions .

What expression systems are most effective for producing recombinant Prochlorococcus marinus atpD?

For effective expression of recombinant Prochlorococcus marinus atpD, E. coli-based expression systems (particularly BL21(DE3) strains) have proven most reliable when the gene is codon-optimized for E. coli. The optimal methodology involves:

• Cloning the atpD gene into a pET-series vector with an N-terminal His-tag for purification

• Expression at lower temperatures (16-20°C) after IPTG induction at OD₆₀₀ of 0.6-0.8

• Supplementation with additional chaperones (GroEL/GroES) to enhance proper folding

• Cell lysis under native conditions using buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10% glycerol

This approach typically yields 3-5 mg of purified protein per liter of culture. Alternative expression systems in cyanobacteria such as Synechocystis sp. PCC 6803 can be considered when native post-translational modifications are critical, though with lower yields .

What are the most effective purification methods for recombinant Prochlorococcus marinus atpD?

The most effective purification protocol for recombinant Prochlorococcus marinus atpD involves a multi-step approach:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with an imidazole gradient (20-250 mM)

• Intermediate Purification: Ion exchange chromatography using a Q-Sepharose column with a NaCl gradient (50-500 mM)

• Polishing Step: Size exclusion chromatography using a Superdex 200 column in buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 5% glycerol

This protocol typically achieves >95% purity as determined by SDS-PAGE. For functional studies, it's crucial to verify that the purified protein retains its native conformation using circular dichroism spectroscopy to analyze secondary structure elements. Similar approaches have been successfully applied for purification of ATP synthase components from other cyanobacteria .

What assays can be used to confirm the functionality of purified recombinant atpD?

To confirm functionality of purified recombinant atpD, researchers should employ multiple complementary assays:

• ATP Hydrolysis Activity Assay: Measure inorganic phosphate release using malachite green or enzyme-coupled assays. Typical wild-type activity ranges from 2-5 μmol Pi/min/mg protein.

• Nucleotide Binding Analysis: Determine binding constants using fluorescent ATP analogs or isothermal titration calorimetry.

• Assembly Verification: Assess interaction with other ATP synthase subunits via:

  • Pull-down assays with tagged partner proteins

  • Native PAGE analysis

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

• Inhibitor Sensitivity Testing: Measure response to known ATP synthase inhibitors like DCCD (dicyclohexylcarbodiimide), which should reduce activity by 60-70% at 20 μM concentration .

Complete functional validation requires reconstituting the beta subunit with other ATP synthase components to form the F₁ complex and measuring ATP synthesis activity.

How does the structure of Prochlorococcus marinus atpD compare to that of other cyanobacteria?

Prochlorococcus marinus atpD shares approximately 70-80% sequence identity with ATP synthase beta subunits from other cyanobacteria, with the highest conservation in the nucleotide-binding domain. Key structural differences include:

• N-terminal Region: Prochlorococcus marinus atpD exhibits distinct sequence variations in the N-terminal region compared to other cyanobacteria, potentially affecting interactions with regulatory proteins.

• Catalytic Site: While the catalytic residues are highly conserved, subtle differences in surrounding amino acids may influence catalytic efficiency under different environmental conditions.

• Interface Regions: Amino acid substitutions at subunit interfaces likely reflect adaptations to Prochlorococcus' unique environmental niche, particularly its ability to thrive in low-nutrient oceanic environments.

Homology modeling based on available crystal structures suggests that these differences primarily affect surface-exposed regions while maintaining the core catalytic architecture. This structural conservation reflects the essential nature of ATP synthesis across cyanobacterial lineages despite their ecological diversification .

What are the known interactions between atpD and regulatory proteins in Prochlorococcus marinus?

Research indicates several important interactions between atpD and regulatory proteins in Prochlorococcus marinus:

• AtpΘ Interaction: Similar to other cyanobacteria, Prochlorococcus marinus atpD likely interacts with AtpΘ, an important regulator that inhibits ATP hydrolysis activity under darkness. This interaction has been confirmed in related cyanobacteria through co-immunoprecipitation and far Western blot analyses, showing binding to the beta subunit (atpD) alongside stronger associations with subunits a and c .

• Membrane Association: The ATP synthase complex in Prochlorococcus, including atpD, associates with thylakoid membranes in a way that is responsive to light conditions, suggesting interactions with membrane-associated regulatory proteins.

• Cross-Complex Regulation: Evidence from proteomic studies suggests potential interactions between ATP synthase components and photosynthetic complexes, facilitating energy balance regulation.

These regulatory interactions are particularly important given Prochlorococcus' need to optimize energy utilization in oligotrophic environments.

How has the atpD gene evolved across Prochlorococcus ecotypes?

Evolutionary analysis of the atpD gene across Prochlorococcus ecotypes reveals important adaptive patterns:

This evolutionary pattern suggests that while ATP synthase function is essential, fine-tuning of atpD has contributed to Prochlorococcus' remarkable adaptation to diverse marine environments.

What phylogenetic methods are most appropriate for analyzing atpD evolution in Prochlorococcus?

For optimal phylogenetic analysis of atpD evolution in Prochlorococcus, the following methodological approach is recommended:

• Sequence Alignment:

  • MUSCLE or MAFFT alignment of codon sequences

  • Manual refinement focusing on conserved domains

  • Filtering with Gblocks to remove poorly aligned regions

• Model Selection:

  • Test multiple nucleotide substitution models (GTR, HKY)

  • Implement codon-based models using CODEML for dN/dS analysis

  • Apply site models (M0, M1a, M2a, M7, M8) to detect selection patterns

• Tree Construction:

  • Maximum likelihood method using RAxML with the GTRCAT model

  • Bootstrap analysis with 100 replicates for branch support

  • Bayesian inference as complementary approach

• Selection Analysis:

  • Branch-site tests comparing null hypothesis (H0) with positive selection hypothesis (H1)

  • Extraction of dN/dS values and log-likelihoods for statistical assessment

  • Use of likelihood ratio tests with chi-squared distribution for significance

This comprehensive approach has successfully identified selection patterns in other Prochlorococcus genes and can be directly applied to atpD evolutionary analysis.

How can site-directed mutagenesis be used to study functional residues in Prochlorococcus marinus atpD?

Site-directed mutagenesis provides powerful insights into functional residues of Prochlorococcus marinus atpD. An effective experimental approach includes:

• Target Selection:

  • Catalytic residues (e.g., βE185, βR189) involved in ATP binding and hydrolysis

  • Interface residues mediating interactions with alpha subunits

  • Conserved residues showing ecotype-specific variations

• Mutagenesis Protocol:

  • QuikChange method with complementary mutagenic primers

  • Verification by Sanger sequencing

  • Expression in parallel with wild-type protein

• Functional Assessment:

  • Comparative ATP hydrolysis assays (wild-type vs. mutant)

  • Nucleotide binding affinity measurements

  • Thermal stability analysis by differential scanning fluorimetry

  • Assembly competence evaluation by pull-down assays

• Data Analysis:

  • Calculate kinetic parameters (Km, kcat) for each mutant

  • Compare relative activities under varying pH and salt concentrations

  • Evaluate structural impacts through circular dichroism

This approach has successfully identified functional residues in ATP synthase complexes from other organisms and can be directly applied to Prochlorococcus marinus atpD .

What approaches are most effective for studying atpD interactions with other ATP synthase subunits?

To effectively study interactions between Prochlorococcus marinus atpD and other ATP synthase subunits, researchers should employ a multi-technique approach:

• In Vitro Reconstitution:

  • Co-expression of atpD with other F₁ subunits (alpha, gamma, delta, epsilon)

  • Affinity tag-based co-purification

  • Activity measurements of reconstituted complexes

• Protein-Protein Interaction Analysis:

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Chemical cross-linking coupled with mass spectrometry to map interaction interfaces

• Fluorescence-Based Techniques:

  • Förster resonance energy transfer (FRET) with fluorescently labeled subunits

  • Fluorescence correlation spectroscopy to measure complex formation

• Structural Approaches:

  • Cryo-electron microscopy of reconstituted complexes

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

These methodologies have proven effective in studying ATP synthase subunit interactions in related organisms and can be adapted for Prochlorococcus marinus. For example, FLAG-tagged components have successfully identified interactions between ATP synthase subunits and regulatory proteins like AtpΘ .

How can recombinant Prochlorococcus marinus atpD be used to study adaptation to environmental stressors?

Recombinant Prochlorococcus marinus atpD serves as an excellent model for studying adaptation to environmental stressors through these methodological approaches:

• Comparative Ecotype Analysis:

  • Express and purify atpD from different Prochlorococcus ecotypes (HL vs. LL)

  • Compare enzymatic parameters under varying conditions (temperature, pH, salinity)

  • Correlate differences to the native ecological niches of source organisms

• Stress Response Characterization:

  • Measure activity under simulated environmental stressors (heat, oxidative stress)

  • Compare kinetic parameters (Km, Vmax) under standard vs. stress conditions

  • Determine thermal stability profiles using differential scanning fluorimetry

• Chimeric Protein Studies:

  • Create chimeric proteins with domains from different ecotypes

  • Map functional differences to specific structural regions

  • Identify critical residues mediating stress adaptation

• Heterologous Complementation:

  • Express Prochlorococcus atpD variants in model cyanobacteria

  • Test growth and photosynthetic efficiency under different light regimes

  • Measure ATP synthesis rates in vivo under environmental stress conditions

This approach provides insights into how ATP synthase adaptations contribute to Prochlorococcus' ecological success across diverse marine environments and helps understand the molecular basis of stress response in this globally important primary producer .

What techniques can be used to investigate the role of atpD in energy balance during light/dark transitions?

Investigating atpD's role in energy balance during light/dark transitions requires sophisticated techniques focusing on dynamic regulation:

• Real-time Activity Monitoring:

  • Develop luciferase-based ATP sensing systems in reconstituted membranes

  • Monitor ATP synthesis/hydrolysis transitions during light shifts

  • Correlate activity changes with regulatory protein interactions

• Inhibitor Studies:

  • Use specific inhibitors like DCCD to block different aspects of ATP synthase function

  • Compare inhibition patterns between light and dark conditions

  • Quantify the relative contribution of ATP synthase to cellular energy balance

• Regulatory Interaction Analysis:

  • Investigate AtpΘ binding to atpD under light/dark conditions

  • Measure the inhibitory effects on ATPase activity in membrane preparations

  • Compare wild-type vs. regulatory mutants' response to darkness

• Membrane Potential Measurements:

  • Use fluorescent probes to monitor membrane potential during light/dark transitions

  • Correlate potential changes with ATP synthase activity

  • Determine the threshold for switching between synthesis and hydrolysis modes

Experimental evidence from related cyanobacteria demonstrates that ATP synthase inhibition is critical during darkness to prevent wasteful ATP hydrolysis. For example, membrane fractions from wild-type cells show significantly higher ATPase activity when grown in light compared to dark conditions, while knockout strains lacking regulatory elements like AtpΘ show no significant difference between light and dark conditions .

What are common challenges in expressing soluble Prochlorococcus marinus atpD and how can they be overcome?

Researchers commonly encounter several challenges when expressing soluble Prochlorococcus marinus atpD, which can be addressed through these specific approaches:

• Inclusion Body Formation:

  • Reduce expression temperature to 16°C

  • Decrease IPTG concentration to 0.1-0.2 mM

  • Co-express with molecular chaperones (GroEL/GroES)

  • Use fusion tags like SUMO or MBP to enhance solubility

• Protein Instability:

  • Add stabilizing agents (glycerol 5-10%, low concentrations of ATP)

  • Optimize buffer conditions (150-300 mM NaCl, pH 7.5-8.0)

  • Include protease inhibitors throughout purification

  • Process samples rapidly at 4°C

• Low Expression Yields:

  • Optimize codon usage for expression host

  • Test multiple expression vectors with different promoter strengths

  • Screen multiple E. coli strains (BL21, Rosetta, Arctic Express)

  • Consider auto-induction media for gradual protein expression

• Protein Misfolding:

  • Implement step-wise refolding procedures if purifying from inclusion bodies

  • Use pulse-renaturation techniques with controlled dilution

  • Add nucleotides during refolding to stabilize native conformation

These approaches have been successfully implemented for expressing other ATP synthase components, with typical yields increasing from <1 mg/L to 3-5 mg/L of properly folded protein .

How can researchers accurately measure the interaction between atpD and potential inhibitors?

For accurate measurement of interactions between atpD and potential inhibitors, researchers should implement these methodological approaches:

• Enzymatic Inhibition Assays:

  • Measure ATP hydrolysis with the malachite green assay in the presence of inhibitors

  • Determine IC₅₀ values through dose-response curves

  • Calculate Ki values using appropriate enzyme inhibition models

  • Compare inhibitory effects on isolated atpD versus assembled F₁ complexes

• Binding Affinity Determination:

  • Microscale thermophoresis (MST) for direct binding measurements

  • Isothermal titration calorimetry (ITC) for complete thermodynamic profiles

  • Surface plasmon resonance (SPR) for association/dissociation kinetics

  • Fluorescence-based assays using intrinsic tryptophan fluorescence

• Structural Analysis of Inhibitor Binding:

  • Hydrogen-deuterium exchange mass spectrometry to map binding sites

  • X-ray crystallography of atpD-inhibitor complexes

  • NMR spectroscopy for dynamic interaction analysis

• Comparative Inhibition Analysis:

  • Compare inhibition patterns against established ATP synthase inhibitors like DCCD

  • Test inhibitor combinations to identify synergistic effects

  • Evaluate inhibitor specificity across ATP synthases from different sources