Recombinant Solanum bulbocastanum ATP synthase subunit c, chloroplastic (atpH)

Basic Research Questions

• What is the molecular structure and functional role of ATP synthase subunit c in S. bulbocastanum chloroplasts?

ATP synthase subunit c (atpH) in S. bulbocastanum functions as a critical component of the F0 portion of the chloroplastic ATP synthase complex, which is responsible for ATP production during photosynthesis. The protein consists of 81 amino acids with the following sequence:

"MNPLIAAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV"

This highly hydrophobic protein serves as the lipid-binding component in the membrane-embedded F0 sector. It forms a ring structure in the thylakoid membrane that rotates during proton translocation, driving conformational changes in the F1 sector that catalyze ATP synthesis. The protein's structure is characterized by a transmembrane alpha-helical domain, typical of F-type ATPase subunit c proteins across species.

The importance of ATP synthase components in plant stress responses has been documented in other species, particularly cucumber, where polymorphisms in the beta-subunit (atpB) have been associated with enhanced cold tolerance . Given S. bulbocastanum's remarkable disease resistance properties, the chloroplast ATP synthase components may contribute to cellular energy homeostasis during pathogen stress responses.

• What expression systems yield optimal results for recombinant S. bulbocastanum atpH protein production?

Several expression systems have been successfully employed for recombinant atpH production, with E. coli being the most widely documented. For optimal expression, researchers have utilized multiple vector systems with different fusion tags, as summarized in the following table:

BL21 derivative E. coli cells (T7 Express lysY/Iq) have demonstrated good results for transformation . For difficult-to-express membrane proteins like atpH, co-expression with molecular chaperones significantly improves yield. The pOFXT7KJE3 plasmid, which expresses the chaperone proteins DnaK, DnaJ, and GrpE, has been shown to substantially increase quantities of recombinant proteins that are otherwise toxic or difficult to produce in bacterial systems .

For researchers requiring isotope-labeled protein for structural studies, expression in minimal media with 13C and 15N sources has been successfully employed for other membrane proteins and could be adapted for atpH production .

• What purification strategies are most effective for isolating recombinant atpH protein?

Effective purification of recombinant atpH depends on the expression system and fusion tags employed. The following methodology-focused approaches yield the best results:

Affinity Chromatography (Tag-based first-step purification):

• For MBP-tagged atpH: Use amylose resin affinity chromatography with elution using maltose buffer (10-20 mM maltose). This typically yields >90% purity in a single step .

• For His-tagged atpH: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co2+ resins with imidazole elution (250-300 mM) works effectively.

• For FLAG-tagged atpH: Anti-FLAG antibody resin with competitive elution using FLAG peptide provides high specificity.

Secondary Purification Steps:
For applications requiring higher purity (>95%), a second chromatographic step is recommended:

• Size exclusion chromatography: Separates monomeric atpH from aggregates and contaminants of different molecular sizes

• Ion exchange chromatography: At pH 7.0-8.0, atpH can be separated based on charge properties

Critical Considerations:

• Due to atpH's hydrophobic nature, inclusion of detergents (0.05-0.1% DDM or 0.5-1% CHAPS) in all buffers is essential to prevent aggregation

• For membrane proteins like atpH, purification at 4°C and inclusion of 10-20% glycerol improves stability

• Protease inhibitor cocktails should be included to prevent degradation

The typical yield for recombinant atpH using the MBP-fusion system is 2-5 mg per liter of bacterial culture, with purities of 85-95% after the first chromatographic step .

• How is recombinant atpH protein best characterized to confirm identity and functionality?

Comprehensive characterization of recombinant atpH requires multiple analytical approaches:

For Identity Confirmation:

• SDS-PAGE: Should show a band at approximately 8-9 kDa for the untagged protein or appropriate higher molecular weight for fusion constructs

• Western blotting: Using antibodies against atpH or the fusion tag

• Mass spectrometry: MALDI-TOF or ESI-MS to confirm exact molecular weight

• N-terminal sequencing: To verify the correct start of the protein

For Structural Characterization:

• Circular dichroism (CD) spectroscopy: To assess secondary structure, especially alpha-helical content (expected to be >60% for properly folded atpH)

• FTIR spectroscopy: Complementary approach for secondary structure determination

• Fluorescence spectroscopy: Intrinsic tryptophan fluorescence can indicate folding status

For Functional Assessment:

• Reconstitution into liposomes: To test proton translocation activity

• ATP synthesis assays: When incorporated into ATP synthase complex

• Binding assays: To assess interaction with other ATP synthase subunits

Results should be compared with known parameters for ATP synthase subunit c proteins from related species to confirm proper folding and function of the recombinant protein.

Advanced Research Questions

• How does the genetic polymorphism of S. bulbocastanum atpH compare to other Solanum species, and what evolutionary significance might these variations hold?

Comparative genomic analysis of chloroplast genes across Solanum species reveals significant evolutionary patterns for ATP synthase components. While specific atpH polymorphisms in S. bulbocastanum are not directly detailed in available research, analysis of chloroplast genomes in related species provides valuable insights:

Chloroplast genome sequencing of S. bulbocastanum has uncovered significant genetic diversity when compared to other Solanum species . In the broader Solanaceae family, ATP synthase components show notable evolutionary conservation with specific divergent regions that may correlate with adaptation to different environmental stresses.

Similar studies in Capsicum species have demonstrated that "subunits of ATP synthase (atp) CDSs accumulated 7 unique and common SNPs" among different species/varieties . These polymorphisms often result in amino acid substitutions that can affect protein function or stability.

Particularly significant in evolutionary analysis are changes in amino acid properties that might alter protein function. In Capsicum studies, researchers found that "amino acid changes were detected toward an increase in isoleucine (I)" and other specific substitutions like "alanine (A) was exclusively changed to threonine (T), glycine (G), and aspartate (D)" .

For S. bulbocastanum, its adaptation to high-altitude Mexican environments and remarkable disease resistance suggest selective pressure on energy metabolism genes, potentially including atpH. Comparative analysis of ATP synthase components across wild and cultivated Solanum species may reveal signatures of selection related to stress adaptation.

The distribution of atpH variants could potentially correlate with the geographic distribution of different S. bulbocastanum populations, offering insights into local adaptation processes.

• What methods can be employed to study the potential role of atpH in S. bulbocastanum's disease resistance mechanisms?

While direct evidence linking atpH to disease resistance in S. bulbocastanum is not established, several methodological approaches can be employed to investigate this potential connection:

Genetic Association Studies:

• Sequence atpH from resistant and susceptible S. bulbocastanum accessions to identify polymorphisms

• Perform association analysis between atpH variants and disease resistance phenotypes

• Conduct QTL mapping to determine if atpH co-localizes with resistance loci

Functional Studies:

• Use CRISPR-Cas9 genome editing of atpH in S. bulbocastanum, as demonstrated for other genes

• Generate transgenic S. tuberosum expressing S. bulbocastanum atpH variants

• Challenge edited/transgenic plants with pathogens like Phytophthora infestans to assess resistance

Molecular and Biochemical Analyses:

• Measure ATP synthesis rates in resistant versus susceptible plants during pathogen challenge

• Analyze ROS production in relation to ATP synthase activity during infection

• Use co-immunoprecipitation to identify potential interactions between atpH and known resistance components

S. bulbocastanum's resistance to P. infestans is primarily attributed to R genes (Rpi-blb1, Rpi-blb2, and Rpi-blb3) , but energy metabolism might play a supporting role. Research in cucumber has demonstrated that a single amino acid change in the ATP synthase beta-subunit (atpB) from threonine to arginine confers enhanced cold tolerance , suggesting that similar mechanisms might operate in pathogen resistance.

The unique compound lysophosphatidylcholine 17:1 (LPC17:1) identified on S. bulbocastanum leaf surfaces inhibits P. infestans , but whether atpH influences the production of such defense compounds remains to be investigated.

• What challenges exist in expressing properly folded and functional atpH in heterologous systems, and how can they be overcome?

Expression of properly folded recombinant atpH presents several technical challenges due to its nature as a membrane protein:

Common Challenges and Solutions:

Research has demonstrated that co-expression with molecular chaperones using the pOFXT7KJE3 plasmid "has been shown to substantially increase quantities of recombinant proteins which are toxic or otherwise difficult to produce" .

For structural studies, incorporation of the protein into nanodiscs or liposomes after purification can help maintain native conformation. Additionally, screening multiple detergents (DDM, LMNG, CHAPS) is critical for identifying optimal solubilization conditions.

Recent advances in genetic code expansion, as described for other challenging proteins, offer potential approaches for incorporating non-canonical amino acids to aid in structural analysis of atpH .

• How can isotope labeling techniques be applied to recombinant atpH for structural studies?

Isotope labeling of recombinant atpH enables sophisticated structural studies using NMR spectroscopy and other techniques. The following methodological approach is recommended:

Expression System Optimization for Isotope Labeling:

• Use E. coli BL21(DE3) or its derivatives with T7 expression system

• Employ a two-stage culture protocol:

  • Initial growth in rich medium to achieve high cell density

  • Transfer to minimal medium containing isotope sources for induction

Labeling Methods and Media Composition:

• For uniform 15N labeling:

  • M9 minimal medium with 15NH4Cl (1 g/L) as sole nitrogen source

  • Supplement with trace elements and vitamins

• For uniform 13C labeling:

  • Use 13C-glucose (2-4 g/L) as sole carbon source

• For dual 13C/15N labeling:

  • Combine both labeled sources in minimal medium

Specialized Techniques for Membrane Protein Labeling:

• Selective amino acid labeling:

  • Supplement minimal medium with specifically labeled amino acids

  • Useful for focusing on regions of interest in atpH

• Segmental labeling:

  • For studying specific domains independently

  • Particularly valuable for the hydrophobic transmembrane region

Purification Considerations for Labeled Proteins:

• Maintain identical purification protocols to unlabeled protein

• Consider deuterated detergents to reduce background signals in NMR

• Verify incorporation efficiency by mass spectrometry (>95% incorporation is optimal)

Recent advancements in labeling membrane proteins, as demonstrated in other studies, show that combining selective deuteration with 13C/15N labeling can significantly improve spectral quality for proteins like atpH .

The successfully labeled atpH can be used for various structural studies, including solution NMR, solid-state NMR in membrane mimetics, and X-ray crystallography with anomalous scattering techniques.