Recombinant Pavlova lutherii ATP synthase subunit c, chloroplastic (atpH)

Basic Research Questions

• What is Pavlova lutherii ATP synthase subunit c and what role does it play in cellular energy metabolism?

  ATP synthase subunit c (atpH) in Pavlova lutherii is a membrane-embedded component of the F0 sector of the chloroplastic ATP synthase complex. This 83-amino acid protein (MNPIISAASVIAAGLSVGLAAIGPGIGQGSAAGQALEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALSLLFANPFTAS) forms the c-ring structure within the F0 sector that facilitates proton translocation across the thylakoid membrane . The protein functions as part of the rotary mechanism that couples proton movement to ATP synthesis during photosynthesis.

  As a key component of the ATP synthase complex, atpH contributes to energy conversion processes that are essential for cellular functions in this microalga. The protein is characterized by its hydrophobic nature and ability to organize into oligomeric ring structures, creating the proton channel necessary for ATP production. Its activity is directly linked to the organism's ability to harvest light energy and convert it into chemical energy.

• How is Pavlova lutherii taxonomically classified, and why has it been renamed to Diacronema lutheri?

  Pavlova lutherii has been reclassified as Diacronema lutheri based on phylogenetic analyses. It belongs to:

  • Domain: Eukaryota

  • Kingdom: Chromista

  • Phylum: Haptophyta

  • Class: Pavlovophyceae

  • Order: Pavlovales

  • Family: Pavlovaceae

  • Genus: Diacronema (formerly Pavlova)

  • Species: D. lutheri (formerly P. lutheri)

  The reclassification reflects evolutionary relationships revealed through genomic studies. The Pavlovales invariably occupy the outer branches of haptophyte phylogenies, at considerable evolutionary distance from coccolithophores and other haptophyte clades . This taxonomic revision has been confirmed through mitochondrial, plastid, and 18S sequence analyses, although both names remain in use in scientific literature .

• What are the structural characteristics of recombinant Pavlova lutherii ATP synthase subunit c?

  The recombinant Pavlova lutherii ATP synthase subunit c (atpH) protein has several notable structural characteristics:

  • Length: 83 amino acids (full-length protein)

  • N-terminal His-tag addition for purification purposes

  • Hydrophobic transmembrane domains that anchor the protein in lipid bilayers

  • Conserved carboxyl group essential for proton binding and transport

  • Secondary structure predominantly comprising alpha-helical segments that span the membrane

  The amino acid sequence (MNPIISAASVIAAGLSVGLAAIGPGIGQGSAAGQALEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALSLLFANPFTAS) reveals a high proportion of hydrophobic residues, consistent with its membrane-embedded nature . The protein's compact structure reflects evolutionary optimization for its role in the ATP synthase complex, with functional domains preserved across diverse species while allowing for organism-specific adaptations.

Advanced Research Questions

• How does the high GC content of the Diacronema lutheri genome affect expression strategies for recombinant atpH production?

  The exceptionally high GC content (73.25%) of the Diacronema lutheri genome presents significant challenges for heterologous expression of its genes, including atpH . This GC skew affects several aspects of recombinant protein production:

  Challenge	Impact	Mitigation Strategy
  Codon bias incompatibility	Reduced translation efficiency in host organisms	Codon optimization for expression host
  Secondary structure formation in mRNA	Impaired translation initiation	Modification of 5' regions to reduce strong secondary structures
  PCR amplification difficulties	Lower fidelity and yield	Use of specialized polymerases and GC-optimized PCR conditions
  Transcriptional challenges	Altered promoter recognition	Selection of appropriate promoters for high-GC templates

  Researchers typically address these challenges through comprehensive codon optimization when designing synthetic genes for recombinant expression. For Diacronema lutheri atpH, adapting the coding sequence to E. coli's preferred codon usage while maintaining the amino acid sequence is essential for successful expression . This optimization process must carefully balance GC content reduction while preserving important regulatory elements that affect protein folding and expression efficiency.

• What analytical techniques are most effective for studying the integration of Pavlova lutherii atpH into membrane systems?

  Several advanced analytical techniques are particularly valuable for studying membrane integration of Pavlova lutherii atpH:

  • Circular Dichroism (CD) Spectroscopy: For analyzing secondary structure content and conformational changes upon membrane insertion

  • Fluorescence Resonance Energy Transfer (FRET): To monitor protein-protein interactions within the ATP synthase complex

  • Cryo-Electron Microscopy: For visualization of the c-ring architecture in near-native conditions

  • Solid-State NMR: To determine structural details of membrane-embedded segments

  • Hydrogen-Deuterium Exchange Mass Spectrometry: For probing solvent-accessible regions and conformational dynamics

  • Surface Plasmon Resonance: To quantify binding kinetics with other ATP synthase subunits

  • Reconstitution into Liposomes: To assess functional properties in defined membrane environments

  When studying Pavlova lutherii atpH, researchers should consider the protein's small size (83 amino acids) and highly hydrophobic nature, which may require specialized approaches for solubilization and sample preparation . Combining structural analysis with functional assays (e.g., proton translocation measurements) provides comprehensive insights into how this protein integrates into and functions within membrane systems.

• How can researchers validate the functionality of recombinant Pavlova lutherii atpH protein?

  Validating the functionality of recombinant Pavlova lutherii atpH protein requires a multi-faceted approach:

  1. Proton Translocation Assays: Reconstitute the protein into liposomes containing pH-sensitive fluorescent dyes to monitor proton movement

  2. ATP Synthesis Measurements: Incorporate atpH into minimal reconstituted ATP synthase systems to measure ATP production rates

  3. Oligomeric State Analysis:

     • Blue-native PAGE to verify assembly into c-rings

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

  4. Binding Studies:

     • Isothermal titration calorimetry (ITC) to quantify interactions with other subunits

     • Pull-down assays with other ATP synthase components to verify complex formation

  5. Mutagenesis Validation: Compare wild-type activity with site-directed mutants of key functional residues

  6. Biophysical Characterization: Circular dichroism to confirm proper secondary structure formation

  A critical consideration is maintaining the hydrophobic protein in a native-like membrane environment or suitable detergent micelles throughout these analyses to preserve its structural integrity and function . Complete validation typically requires comparison with native ATP synthase c-subunit activity whenever possible.

Methodological Considerations

• What are the optimal conditions for solubilizing and stabilizing recombinant Pavlova lutherii atpH protein?

  Optimal solubilization and stabilization of recombinant Pavlova lutherii atpH requires careful consideration of its hydrophobic nature. Based on experiences with similar membrane proteins, the following approaches are recommended:

  Solubilization Methods:

  • Mild detergents: n-Dodecyl β-D-maltoside (DDM) or n-Decyl-β-D-maltopyranoside (DM) at 1-2% (w/v)

  • Newer amphipathic agents: LMNG (Lauryl Maltose Neopentyl Glycol) or SMA (Styrene Maleic Acid) copolymers

  • Reconstitution into nanodiscs with MSP (Membrane Scaffold Protein) and defined lipids

  Stabilization Strategies:

  • Buffer composition: 20-50 mM Tris or phosphate buffer (pH 7.5-8.0) with 100-150 mM NaCl

  • Addition of glycerol (10-25%) to prevent aggregation

  • Inclusion of specific lipids that interact with ATP synthase (e.g., cardiolipin)

  Storage Recommendations:

  • Aliquot purified protein and store at -80°C to avoid freeze-thaw cycles

  • For working stocks, store at 4°C for up to one week in buffer containing 6% trehalose

  • Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL with 5-50% glycerol

  The choice of method should be dictated by the downstream applications. For structural studies, detergent screening is essential to identify conditions that maintain the native oligomeric state while providing sufficient protein stability.

• What are the recommended procedures for expression and purification of recombinant Pavlova lutherii atpH protein?

  Expressing and purifying recombinant Pavlova lutherii atpH protein requires specialized protocols designed for membrane proteins:

  Expression System Selection:

  • E. coli BL21(DE3) is commonly used for His-tagged constructs

  • Consider C41(DE3) or C43(DE3) strains specifically engineered for membrane protein expression

  • Expression temperature should be lowered to 16-20°C to allow proper membrane insertion

  Induction Protocol:

  • Use low IPTG concentrations (0.1-0.5 mM) for gentle induction

  • Extend expression time to 16-24 hours at reduced temperature

  Purification Workflow:

  1. Membrane isolation by ultracentrifugation following cell lysis

  2. Solubilization in appropriate detergent (2-4 hours at 4°C)

  3. IMAC purification using Ni-NTA resin to capture His-tagged protein

  4. Size exclusion chromatography to achieve final purity

  Quality Control:

  • SDS-PAGE to confirm >90% purity

  • Western blotting with anti-His antibodies to verify identity

  • Mass spectrometry to confirm exact molecular weight

  • Circular dichroism to assess secondary structure integrity

  Throughout the purification process, maintaining the cold chain and working quickly is essential to prevent protein aggregation or degradation. The final preparation should be assessed for both purity and functional activity before use in downstream applications.

• How can site-directed mutagenesis be applied to study the structure-function relationship of Pavlova lutherii atpH?

  Site-directed mutagenesis offers powerful insights into structure-function relationships of Pavlova lutherii atpH. Key approaches include:

  Strategic Residue Targeting:

  • Conserved glutamate/aspartate residues in the ion-binding site

  • Residues at subunit interfaces involved in c-ring assembly

  • Amino acids participating in interactions with other ATP synthase components

  • Hydrophobic residues that anchor the protein in the membrane

  Mutation Design Principles:

  • Conservative substitutions to preserve structure while altering specific properties

  • Charge-altering mutations to probe electrostatic interactions

  • Introduction of reporter groups (e.g., cysteine residues for labeling)

  • Creation of chimeric constructs with c-subunits from other species

  Functional Analysis Framework:

  1. Express and purify wild-type and mutant proteins in parallel

  2. Compare structural properties (oligomerization, thermal stability)

  3. Assess proton translocation capabilities in reconstituted systems

  4. Measure effects on ATP synthesis rates when incorporated into the ATP synthase complex

  Example Mutation Panel for Pavlova lutherii atpH:

  Mutation Type	Target Residues	Expected Effect	Analysis Method
  Ion coordination	E56A, D61N	Altered proton binding	pH-dependent spectroscopy
  Helix-helix interaction	L40A, I44A	Disrupted c-ring assembly	Blue-native PAGE
  Lipid interaction	F77W	Modified membrane association	Fluorescence spectroscopy
  Conformational probe	S22C	Site-specific labeling	FRET analysis

  This systematic mutagenesis approach can reveal critical insights into how the relatively small (83 amino acids) but functionally essential atpH protein contributes to ATP synthase activity in Pavlova lutherii .

• What comparative genomic insights can be gained from studying Pavlova lutherii atpH in relation to other haptophyte species?

  Comparative genomic analysis of Pavlova lutherii atpH provides valuable evolutionary insights:

  Genomic Context:

  • The Diacronema lutheri genome is remarkably compact (43.503 Mb) compared to other haptophytes

  • It contains 14,446 protein-coding genes with high GC content (73.25%)

  • This contrasts with larger haptophyte genomes like Emiliania huxleyi (155.931 Mb)

  Evolutionary Conservation:

  • ATP synthase components show varying degrees of conservation across haptophyte lineages

  • The c-subunit (atpH) is typically among the most conserved due to functional constraints

  • Sequence variations occur primarily in loop regions while transmembrane domains remain conserved

  Phylogenetic Positioning:

  • Pavlovales occupy distinct outer branches in haptophyte phylogenies

  • Significant evolutionary distance exists between Pavlova/Diacronema and coccolithophores

  Functional Adaptations:

  • Sequence variations may reflect adaptation to different environmental conditions

  • Lipid composition differences between species influence ATP synthase function

  • Pavlova lutherii's unusual lipid metabolism (including unique betaine lipids) may interact with ATP synthase structure

  These comparative analyses provide a framework for understanding how selective pressures have shaped ATP synthase evolution in different haptophyte lineages, potentially revealing adaptation mechanisms to diverse marine environments.

Technical Applications and Troubleshooting

• What are common technical challenges in working with recombinant Pavlova lutherii atpH and how can they be addressed?

  Several technical challenges are commonly encountered when working with recombinant Pavlova lutherii atpH:

  Challenge	Cause	Solution
  Poor expression yield	Membrane protein toxicity, codon bias	Use specialized expression strains (C41/C43), optimize codons for E. coli
  Protein aggregation	Hydrophobic interactions, improper folding	Express at lower temperatures (16-20°C), include membrane-mimicking agents
  Insufficient purity	Non-specific binding to purification resins	Optimize imidazole concentration in wash buffers, add secondary purification step
  Loss of activity	Detergent-induced denaturation	Screen detergent panel, consider lipid nanodisc reconstitution
  Oligomerization variability	Destabilization of c-ring structure	Chemical crosslinking to stabilize native oligomeric state
  Analytical interference	Detergent micelles affecting size analysis	Use detergent-compatible analytical methods (SEC-MALS with detergent correction)

  A systematic approach to optimization is essential, with careful documentation of conditions that successfully maintain protein stability. When reconstituting the protein from lyophilized form, adding glycerol (5-50% final concentration) and aliquoting to avoid freeze-thaw cycles is strongly recommended . For particularly challenging analyses, newer membrane-mimetic systems like styrene-maleic acid lipid particles (SMALPs) may preserve native lipid interactions better than traditional detergent solubilization.

• How can researchers design experiments to investigate the lipid-protein interactions of Pavlova lutherii atpH?

  Investigating lipid-protein interactions of Pavlova lutherii atpH requires specialized experimental approaches:

  Reconstitution Studies:

  • Systematic reconstitution into liposomes with defined lipid compositions

  • Comparison of protein activity in lipid environments mimicking native Pavlova lutherii membranes versus standard phospholipids

  • Special consideration for the unique betaine lipids (DGCC and DGTA) found in Pavlova species

  Biophysical Approaches:

  • Differential scanning calorimetry to measure thermal stability in different lipid environments

  • Electron paramagnetic resonance (EPR) with spin-labeled lipids to measure binding affinities

  • Solid-state NMR to detect specific lipid-protein contacts

  Molecular Dynamics Simulations:

  • In silico modeling of atpH-lipid interactions with different membrane compositions

  • Calculation of binding energies with specific lipid types

  • Prediction of lipid binding sites on the protein surface

  Functional Correlation:

  • Measurement of proton translocation efficiency in different lipid environments

  • Correlation of lipid binding strength with functional parameters

  Since Pavlova lutherii/Diacronema lutheri synthesizes unusual lipids including betaine lipids and unique dihydroxylated sterols (pavlovols) , testing how these specific lipids interact with atpH could provide insights into evolutionary adaptations of ATP synthase in this organism. The lipid-binding characteristics mentioned in the protein's alternative name (Lipid-binding protein) suggest particular importance of these interactions.

• What are the considerations for designing structure-function studies comparing ATP synthase components across different haptophyte species?

  Designing comparative structure-function studies of ATP synthase across haptophyte species requires careful consideration of several factors:

  Experimental Design Elements:

  1. Species Selection Criteria:

     • Include representatives from major haptophyte lineages (Pavlovales, Isochrysidales, Coccolithales)

     • Compare species with different genome sizes and GC content

     • Consider ecological adaptations (coastal vs. oceanic, temperature preferences)

  2. Standardized Expression Systems:

     • Use identical expression vectors and hosts for all orthologous genes

     • Apply consistent codon optimization strategies across species

     • Maintain identical purification tags and protocols

  3. Comparative Analysis Framework:

     • Structural comparisons (protein size, oligomeric state, stability)

     • Functional parameters (proton translocation rates, ATP synthesis efficiency)

     • Lipid interaction profiles (binding preferences, effects on activity)

  4. Integration with Genomic Data:

     • Correlate functional differences with genomic features

     • Consider the context of gene location and organization

     • Examine evolutionary rate variation in ATP synthase components

  Case Study: Comparing Diacronema lutheri with Emiliania huxleyi

  Feature	Diacronema lutheri	Emiliania huxleyi	Analytical Approach
  Genome size	43.503 Mb	155.931 Mb	Genomic context analysis
  GC content	73.25%	65.67%	Codon usage optimization
  Membrane composition	Unique betaine lipids	Standard phospholipids	Lipid-dependent activity assays
  Ecological niche	Coastal environments	Oceanic, bloom-forming	Thermal/pH stability comparisons

  This comparative approach can reveal how evolutionary pressures have shaped ATP synthase components in different haptophyte lineages, potentially uncovering adaptation mechanisms that could inform biotechnological applications.

• How can researchers optimize protocols for crystallization or cryo-EM studies of Pavlova lutherii ATP synthase components?

  Optimizing structural studies of Pavlova lutherii ATP synthase components requires specialized approaches for membrane proteins:

  Sample Preparation Considerations:

  1. Protein Engineering Strategies:

     • Truncation of flexible regions to enhance conformational homogeneity

     • Addition of crystallization chaperones (e.g., antibody fragments, designed ankyrin repeat proteins)

     • Introduction of thermostabilizing mutations

  2. Detergent Screening Protocol:

     • Systematic testing of detergent types (maltoside, glucoside, and neopentyl glycol classes)

     • Detergent concentration optimization to maintain minimum required for solubility

     • Mixed detergent approaches to improve crystal contacts

  3. Lipid Supplementation:

     • Addition of specific lipids known to interact with ATP synthase

     • Testing effects of betaine lipids unique to Pavlova species

     • Bicelle or lipidic cubic phase approaches for crystallization

  Cryo-EM Specific Considerations:

  • Grid preparation optimization (blotting times, ice thickness)

  • Testing of various support films (graphene oxide, gold, carbon)

  • Use of detergent alternatives (SMALPs, nanodiscs, amphipols)

  Crystallization Screening Strategy:

  • Vapor diffusion with specialized membrane protein screens

  • LCP (Lipidic Cubic Phase) crystallization for highly hydrophobic components

  • Micro-seeding to improve crystal quality

  Practical Workflow:

  1. Initial purification and quality assessment via SEC-MALS

  2. Thermal stability screening across conditions (differential scanning fluorimetry)

  3. Small-scale crystallization or cryo-EM grid preparation screening

  4. Optimization of promising conditions

  The small size of atpH (83 amino acids) presents challenges for cryo-EM studies as an isolated protein, so researchers may need to focus on capturing larger ATP synthase subcomplexes that include atpH or utilize crystallographic approaches for the isolated subunit.

• What are the most effective approaches for studying the role of Pavlova lutherii atpH in energy metabolism and lipid biosynthesis pathways?

  Investigating the role of Pavlova lutherii atpH in energy metabolism and lipid biosynthesis requires integrative approaches:

  Metabolic Flux Analysis:

  • Stable isotope labeling to trace carbon flow through ATP-dependent pathways

  • Comparative analysis of wild-type versus atpH-modified strains

  • Correlation of ATP production rates with lipid biosynthesis activities

  Gene Expression Manipulation:

  • Development of genetic tools for Pavlova lutherii (challenging due to high GC content)

  • Controlled modulation of atpH expression levels

  • Analysis of compensatory responses in ATP synthase complex

  Systems Biology Integration:

  • Correlation of atpH activity with transcriptomic profiles

  • Integration with the 14,446 protein-coding genes identified in the genome

  • Network analysis of energy-lipid metabolism connections

  Environmental Response Studies:

  • Analysis of atpH regulation under different growth conditions

  • Examination of ATP production efficiency during lipid accumulation phases

  • Investigation of energy partitioning during stress responses

  Diacronema lutheri is particularly valuable for these studies due to its established role in aquaculture and as a key organism for studying microalgal lipid biosynthesis . Its unique ability to produce both eicosapentaenoic acid (20:5 n−3) and docosahexaenoic acid (22:6 n−3) makes it an excellent model for investigating the energetic requirements of complex lipid biosynthesis pathways. The compact genome and streamlined metabolism of this organism provide advantages for systems-level studies of energy-lipid relationships.