Recombinant Ranunculus macranthus ATP synthase subunit c, chloroplastic (atpH)

What is ATP synthase subunit c, chloroplastic (atpH) in Ranunculus macranthus?

ATP synthase subunit c, chloroplastic (atpH) in Ranunculus macranthus is a critical protein component of the chloroplast ATP synthase complex involved in energy production during photosynthesis . This protein belongs to the F-type ATPase subunit c family and functions within the F0 sector of the ATP synthase machinery . The protein consists of 81 amino acids with the sequence: MNPLISAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV . As part of the ATP synthase complex, it facilitates proton movement across the thylakoid membrane, contributing to the electrochemical gradient necessary for ATP synthesis during photosynthesis . The protein is encoded by the atpH gene located in the chloroplast genome, typically positioned between atpF and atpI genes in the large single-copy (LSC) region in most Ranunculus species .

What experimental techniques are commonly used to express and purify recombinant atpH protein?

The expression and purification of recombinant Ranunculus macranthus ATP synthase subunit c involve several specialized techniques optimized for chloroplast membrane proteins. Initially, researchers clone the atpH gene into appropriate expression vectors, typically incorporating affinity tags to facilitate purification . Given that atpH is a small, hydrophobic membrane protein (81 amino acids), expression systems that accommodate membrane proteins are preferred, such as E. coli strains specifically engineered for membrane protein expression .

The purification process involves:

• Cell lysis under conditions that preserve protein structure

• Membrane isolation via differential centrifugation

• Solubilization using mild detergents (typically CHAPS or n-dodecyl-β-D-maltoside)

• Affinity chromatography utilizing the incorporated tag

• Size-exclusion chromatography for final purification

For structural studies, researchers may employ techniques such as circular dichroism spectroscopy to assess secondary structure or more advanced methods like NMR spectroscopy, given the protein's relatively small size. The purified protein is typically stored in a Tris-based buffer with 50% glycerol at -20°C to maintain stability, with extended storage recommended at -80°C . For functional studies, reconstitution into liposomes may be necessary to assess proton transport capabilities.

How does the atpH gene rearrangement in different Ranunculus species impact phylogenetic analyses?

The atpH gene rearrangement observed in select Ranunculus species represents a significant molecular marker for phylogenetic analyses. In R. austro-oreganus and R. occidentalis, the atpH gene has translocated from its typical position in the LSC region to between ycf1 and trnN-GUU in the SSC region, which is approximately 11.5 kb away from its conventional location . This substantial genomic rearrangement correlates with an expansion of the SSC region in these species to approximately 21,249 bp and 21,269 bp, respectively, compared to the standard 18,909 bp in R. japonicus and R. macranthus .

These genomic rearrangements serve as powerful phylogenetic markers for several reasons:

• Such structural changes are relatively rare events in chloroplast evolution

• They occur at lower frequencies than nucleotide substitutions, reducing homoplasy

• They provide strong evidence for common ancestry when shared by multiple species

For comprehensive phylogenetic analyses, researchers should implement a multi-faceted approach:

• Include both gene sequence data and structural rearrangement information

• Apply software tools like IRscope to accurately detect IR expansion/contraction events

• Employ sliding window analysis with DnaSP v.5 to quantify nucleotide variability across the genome

These structural variations offer valuable insights into the evolutionary relationships within Ranunculus, potentially resolving phylogenetic ambiguities that sequence data alone cannot address. The fact that only two species within the analyzed group show this rearrangement strongly suggests their close evolutionary relationship and potential divergence from other Ranunculus species .

What methods can be used to investigate positive selection in the atpH gene across Ranunculus species?

The PAML package (Phylogenetic Analysis by Maximum Likelihood) represents the gold standard for detecting positive selection. Specifically, researchers should implement:

• CODEML analysis with the branch model (model = 2; NSsites = 0) to estimate the dN:dS ratio (ω) across lineages

• Comparison between foreground (Ranunculeae) and background (outgroup) branches to identify lineage-specific selection

• Statistical validation using chi-square distribution to assess significance of results

• Bayes Empirical Bayes (BEB) method to identify specific amino acid sites under selection with high posterior probability values (P > 0.9)

This analytical framework should be complemented with:

• Extraction of complete coding sequences (CDS) using tools like Geneious Prime

• Codon-based Z-test of selection implemented in MEGA software

• McDonald-Kreitman test to compare intraspecific polymorphism versus interspecific divergence

While studies have identified several chloroplast genes showing positive selection in Ranunculus (including ndhE, ndhF, rpl23, atpF, rps4, and rpoA), specific analysis of atpH selection pressures would provide valuable insights into how its functional constraints and evolutionary dynamics may differ from related genes in the ATP synthase complex . The unique positioning of atpH in certain Ranunculus species further suggests potential selective pressures related to genomic reorganization.

How can structural variations in the atpH protein be systematically analyzed across species?

Systematic analysis of structural variations in the atpH protein across Ranunculus species requires an integrated approach combining sequence analysis, structural prediction, and comparative genomics. The following methodology provides a comprehensive framework:

• Sequence Alignment and Conservation Analysis:

  • Multiple sequence alignment of atpH proteins from diverse Ranunculus species

  • Calculation of conservation scores at each amino acid position

  • Identification of species-specific substitutions versus genus-conserved regions

• Structural Prediction and Modeling:

  • Prediction of transmembrane domains using specialized algorithms (TMHMM, Phobius)

  • 3D structure modeling using homology modeling with templates from related ATP synthase subunit c structures

  • Molecular dynamics simulations to assess structural stability of variant forms

• Functional Domain Analysis:

  • Mapping of essential functional domains for proton transport

  • Assessment of how species-specific variations might impact protein-protein interactions within the ATP synthase complex

  • Evaluation of potential functional consequences of amino acid substitutions

• Association with Genomic Context:

  • Correlation analysis between protein structural variations and the genomic positioning of atpH

  • Investigation of whether species with gene rearrangements (R. austro-oreganus and R. occidentalis) show corresponding protein adaptations

This systematic approach enables researchers to connect sequence-level variations with potential functional and evolutionary implications, particularly in species showing atypical genomic arrangements of the atpH gene. The comprehensive analysis framework facilitates understanding whether structural adaptations in the protein complement the observed genomic rearrangements.

What are the optimal conditions for functional characterization of recombinant atpH protein?

The functional characterization of recombinant Ranunculus macranthus ATP synthase subunit c requires specialized techniques that account for its nature as a membrane protein component of a larger complex. Optimal experimental conditions include:

Reconstitution System Selection:

• Liposome reconstitution using a mixture of phosphatidylcholine and phosphatidic acid (7:3 ratio)

• Nanodiscs stabilized with membrane scaffold proteins for single-particle analysis

• Proteoliposomes with co-reconstituted ATP synthase subunits for complex analysis

Buffer Conditions:

• pH range: 7.2-7.8 (Tris or HEPES buffer)

• Salt concentration: 100-150 mM KCl or NaCl

• Presence of 5-10% glycerol to enhance stability

• Addition of 2-5 mM MgCl₂ as a cofactor

Proton Transport Assays:

• Fluorescence-based assays using pH-sensitive dyes (ACMA or pyranine)

• Membrane potential measurements with voltage-sensitive dyes (Oxonol VI)

• Stopped-flow spectroscopy for kinetic analysis of proton movement

ATP Synthesis Coupling:

• Measurement of ATP synthesis rates using luciferase-based luminescence assays

• Analysis of P:O ratio (ATP synthesized per oxygen consumed) in reconstituted systems

• Assessment of proton conductance using patch-clamp techniques on proteoliposomes

The integration of atpH into the complete ATP synthase complex often requires co-expression or co-reconstitution with other subunits, particularly those of the F₀ sector. Temperature stability analysis is also recommended, as the functional temperature range may provide insights into ecological adaptations of Ranunculus macranthus relative to other species within the genus that show different genomic arrangements of the atpH gene .

What considerations should be made when designing site-directed mutagenesis experiments for atpH functional studies?

When designing site-directed mutagenesis experiments for Ranunculus macranthus atpH functional studies, researchers should incorporate several critical considerations to ensure meaningful results:

Target Site Selection:

• Prioritize highly conserved residues identified through multiple sequence alignments of atpH across Ranunculus species

• Focus on the essential functional motif "GQGTAAGQAVEGIARQPEAEGKIRGTLLL" (residues 26-53) involved in proton translocation

• Investigate species-specific amino acid variations, particularly between species with different genomic arrangements of atpH

Mutation Strategy Matrix:

Experimental Validation Approaches:

• Combine in vitro and in vivo systems to comprehensively assess mutant phenotypes

• Employ complementation studies in ATP synthase-deficient bacterial strains

• Utilize reconstituted proteoliposomes to measure proton transport efficiency

• Implement thermal stability assays to evaluate structural impacts of mutations

Controls and Comparisons:

• Generate parallel mutations in atpH from species with different genomic arrangements (R. austro-oreganus vs. R. macranthus)

• Include subtle mutations (conservative substitutions) as controls for expression/folding effects

• Compare results with homologous mutations in well-characterized ATP synthase subunit c from model organisms

By systematically addressing these considerations, researchers can develop mutagenesis experiments that not only elucidate structure-function relationships in atpH but also potentially reveal evolutionary adaptations associated with the observed genomic rearrangements in certain Ranunculus species.

How does the genomic context of the atpH gene vary across plant families compared to Ranunculus species?

The genomic context of the atpH gene shows notable variations across plant families, with Ranunculus species exhibiting particularly interesting evolutionary patterns. Comparative analysis reveals several distinct organizational patterns:

Standard Arrangement in Most Angiosperms:
In most flowering plants, including many Ranunculus species such as R. macranthus, the atpH gene is positioned between atpF and atpI genes within the large single-copy (LSC) region of the chloroplast genome . This arrangement is considered the ancestral state in angiosperms and is widespread across diverse plant families including Convolvulaceae (as seen in Ipomoea pes-caprae) .

Ranunculus-Specific Variations:
A striking deviation from this pattern occurs in two Ranunculus species: R. austro-oreganus and R. occidentalis, where the atpH gene has translocated to the small single-copy (SSC) region, specifically between ycf1 and trnN-GUU genes . This rearrangement coincides with a significant expansion of the SSC region (approximately 2 kb longer than typical Ranunculus species) .

Comparative Framework Across Plant Families:

The unusual repositioning of atpH in select Ranunculus species represents a valuable case study in chloroplast genome evolution. This rearrangement likely occurred through expansion and contraction of the inverted repeat (IR) regions affecting the IR-SSC boundary . Such structural variations can be detected through comparative genomic tools including mVISTA software implementing LAGAN and Shuffle-LAGAN modes, as well as IRscope for detailed analysis of IR boundary shifts .

What methodologies are most effective for detecting positive selection in chloroplast genes like atpH?

Detecting positive selection in chloroplast genes like atpH requires sophisticated analytical approaches that can distinguish between neutral evolution and adaptive changes. The most effective methodologies combine sequence-based analyses with structural and functional insights:

1. Codon-Based Maximum Likelihood Methods:
The most powerful approach employs the PAML package (Phylogenetic Analysis by Maximum Likelihood), particularly CODEML . This method:

• Calculates the ratio of nonsynonymous to synonymous substitution rates (dN/dS or ω)

• Implements site, branch, and branch-site models to detect selection at different levels

• Uses statistical frameworks like Likelihood Ratio Tests (LRTs) to evaluate significance

For atpH analysis, researchers should implement:

• Branch model (model = 2; NSsites = 0) to compare selection pressures between Ranunculus and outgroups

• Site models to identify specific codons under selection

• Bayes Empirical Bayes (BEB) analysis to calculate posterior probabilities for positively selected sites

2. Population Genetics Approaches:
Complementary methods include:

• McDonald-Kreitman test comparing polymorphism within species versus divergence between species

• Tajima's D and Fu & Li's F* tests to detect departures from neutrality

• Fay and Wu's H to identify selective sweeps

3. Integrated Structural-Functional Analysis:
More sophisticated analyses incorporate:

• Mapping of potentially selected sites onto protein structural models

• Assessment of whether selected sites cluster in functional domains

• Correlation of selection patterns with genomic rearrangements (particularly relevant for atpH in Ranunculus)

4. Comparative Analysis Framework:
For robust results, researchers should:

• Include diverse sampling across the Ranunculus genus and related outgroups

• Extract complete coding sequences using tools like Geneious Prime

• Implement sliding window analysis to identify selection hotspots

• Compare selection patterns between species with different atpH genomic arrangements

Studies on related chloroplast genes have identified several loci under positive selection in Ranunculus (ndhE, ndhF, rpl23, atpF, rps4, and rpoA) . Similar methodologies applied to atpH would reveal whether its distinct genomic arrangements correlate with adaptive molecular evolution.

How can researchers integrate atpH variation data with broader chloroplast genome evolution studies?

Integrating atpH variation data with broader chloroplast genome evolution studies requires a multi-dimensional approach that connects gene-level changes with genome-wide patterns. Researchers can implement the following comprehensive framework:

1. Multi-scale Comparative Genomics:

• Synteny analysis using mVISTA with LAGAN and Shuffle-LAGAN modes to identify structural rearrangements across species

• Detailed IR boundary analysis using IRscope to characterize expansion/contraction events affecting atpH positioning

• Calculation of nucleotide diversity (Pi) using sliding window analysis in DnaSP v.5 to identify evolutionary hotspots

• Integration of genome-wide patterns with atpH-specific variations to determine whether they represent isolated events or coordinated evolutionary changes

2. Phylogenomic Integration:

• Construction of phylogenetic trees using both whole chloroplast genomes and individual genes

• Comparison of tree topologies to identify potential discordances suggestive of horizontal gene transfer or incomplete lineage sorting

• Mapping of atpH rearrangements onto phylogenetic trees to determine their timing and frequency

• Implementation of ancestral state reconstruction to understand the directionality of atpH genomic repositioning events

3. Molecular Evolution Integration:

• Correlation analysis between positive selection patterns and structural rearrangements

• Investigation of whether species with repositioned atpH (R. austro-oreganus and R. occidentalis) show distinctive selection signatures

• Comparison of evolutionary rates between standard and rearranged genomic contexts

4. Functional Implication Analysis:

• Assessment of whether atpH repositioning affects gene expression patterns

• Investigation of potential impacts on ATP synthase assembly and function

• Experimental validation of fitness effects associated with different genomic arrangements

This integrated approach enables researchers to contextualize atpH variations within the broader evolutionary history of chloroplast genomes. The unique rearrangements observed in select Ranunculus species provide an exceptional opportunity to study the mechanisms and consequences of chloroplast genome restructuring . By connecting gene-specific changes with genome-wide patterns, researchers can develop more comprehensive models of chloroplast genome evolution that account for both sequence-level and structural variations.

What are the main challenges in expressing and purifying membrane proteins like atpH for structural studies?

Expressing and purifying membrane proteins like atpH for structural studies presents numerous technical challenges due to their hydrophobic nature and integration within lipid bilayers. These challenges and their solutions include:

1. Expression System Limitations:

• Challenge: Conventional bacterial expression systems often result in toxicity, aggregation, or inclusion body formation due to atpH's hydrophobicity.

• Solutions:

  • Utilize specialized E. coli strains designed for membrane protein expression (C41(DE3), C43(DE3))

  • Implement inducible expression systems with tight regulation

  • Explore cell-free expression systems that allow direct incorporation into artificial membranes

  • Consider fusion partners that enhance solubility (MBP, SUMO) with cleavable linkers

2. Extraction and Solubilization Difficulties:

• Challenge: Harsh detergents can denature membrane proteins, while mild detergents may inadequately solubilize.

• Solutions:

  • Screen detergent panels (DDM, LMNG, CHAPS) at various concentrations

  • Implement systematic detergent stability assays

  • Explore nanodisc technology for detergent-free purification

  • Consider styrene maleic acid copolymer (SMA) for native lipid co-extraction

3. Protein Stability Issues:

• Challenge: ATP synthase subunit c is often unstable when isolated from its complex.

• Solutions:

  • Incorporate stabilizing additives (glycerol, specific lipids)

  • Optimize buffer conditions (pH, ionic strength)

  • Consider co-expression with interacting partners

  • Implement thermostability assays to identify optimal conditions

4. Crystallization Barriers:

• Challenge: Small membrane proteins like atpH (81 amino acids) provide limited hydrophilic surface for crystal contacts.

• Solutions:

  • Explore lipidic cubic phase crystallization methods

  • Consider antibody fragment co-crystallization

  • Implement surface engineering to enhance crystallizability

  • Explore alternative structural determination methods (NMR, cryo-EM)

5. Functional Verification:

• Challenge: Confirming that purified atpH retains native folding and functionality.

• Solutions:

  • Develop proton conductance assays using reconstituted proteoliposomes

  • Implement circular dichroism to verify secondary structure

  • Utilize thermal shift assays to assess stability

  • Consider complementation studies in ATP synthase-deficient strains

By systematically addressing these challenges with appropriate methodological solutions, researchers can successfully express, purify, and characterize the structural properties of atpH, contributing valuable insights into its role within the ATP synthase complex.

How can researchers effectively compare atpH gene arrangements across multiple Ranunculus species?

Effectively comparing atpH gene arrangements across multiple Ranunculus species requires a comprehensive analytical framework that integrates genomic, bioinformatic, and visualization approaches. The following methodology provides a systematic pipeline for such comparative analyses:

1. Sequencing and Assembly Strategy:

• Implement genome skimming approaches using next-generation sequencing (NGS) on platforms like Illumina NovaSeq 6000

• Generate paired-end reads (2 × 150 bp) to ensure adequate coverage of the chloroplast genome

• Extract chloroplast reads by mapping to reference genomes using Bowtie 2

• Assemble complete chloroplast genomes using specialized software like Canu (V2.2)

• Annotate resulting genomes with programs like PGA or GeSeq with manual verification

2. Comparative Genomic Analysis:

• Align complete chloroplast genomes using mVISTA with both LAGAN and Shuffle-LAGAN modes to detect structural variations

• Implement IRscope specifically to visualize and quantify IR expansion/contraction events that may affect atpH positioning

• Calculate nucleotide variability (Pi) using sliding window analysis in DnaSP v.5 to identify evolutionary hotspots across the genome

• Use Geneious Prime for detailed gene-level comparisons and codon usage analysis

3. Visualization and Documentation Framework:

4. Statistical Validation:

• Implement statistical tests to evaluate the significance of observed structural variations

• Assess correlation between genomic rearrangements and other evolutionary patterns

• Calculate the frequency of independent rearrangement events across the phylogeny

This comprehensive approach has successfully identified the unique repositioning of atpH in R. austro-oreganus and R. occidentalis, where it has moved from its typical location in the LSC region (between atpF and atpI) to the SSC region (between ycf1 and trnN-GUU) . Such thorough comparative analysis enables researchers to document patterns of chloroplast genome evolution and identify potential mechanisms driving structural rearrangements.

What emerging technologies show promise for investigating atpH function in photosynthetic organisms?

Several emerging technologies show significant promise for investigating atpH function in photosynthetic organisms, offering unprecedented insights into its structure, dynamics, and interactions within the ATP synthase complex:

1. Advanced Structural Biology Approaches:

• Cryo-Electron Microscopy (Cryo-EM): Enables visualization of the entire ATP synthase complex at near-atomic resolution without crystallization, revealing the native context of atpH within the membrane environment

• Integrative Structural Biology: Combines multiple techniques (X-ray crystallography, NMR, SAXS, computational modeling) to generate comprehensive structural models of atpH in different functional states

• Time-Resolved Serial Femtosecond Crystallography: Captures dynamic structural changes during proton translocation using X-ray free-electron lasers (XFELs)

2. Single-Molecule Techniques:

• Single-Molecule FRET: Monitors real-time conformational changes in individually labeled atpH proteins during ATP synthesis

• High-Speed Atomic Force Microscopy (HS-AFM): Visualizes rotational dynamics of ATP synthase components including the c-ring containing atpH subunits

• Patch-Clamp Electrophysiology: Measures proton conductance through single ATP synthase complexes in nanodiscs or liposomes

3. Advanced Genetic and Genomic Approaches:

• CRISPR-Cas9 Genome Editing in Chloroplasts: Enables precise modification of the native atpH gene in photosynthetic organisms

• Site-Specific Unnatural Amino Acid Incorporation: Allows introduction of biophysical probes at specific positions within atpH

• Long-Read Sequencing Technologies (Oxford Nanopore, PacBio): Facilitates more accurate chloroplast genome assembly to detect complex structural rearrangements affecting atpH

4. Systems Biology Integration:

• Multi-omics Approaches: Integrates transcriptomics, proteomics, and metabolomics to understand atpH function in the broader context of photosynthetic energy metabolism

• In Silico Modeling: Implements molecular dynamics simulations to predict how sequence variations impact proton movement through the c-ring

• Synthetic Biology Redesign: Engineers modified atpH variants to optimize photosynthetic efficiency or adapt to different environmental conditions

How might the unique genomic arrangement of atpH in certain Ranunculus species inform synthetic biology applications?

The unique genomic arrangement of atpH in certain Ranunculus species offers valuable insights that could inform innovative synthetic biology applications, particularly in chloroplast engineering and artificial photosynthetic systems:

1. Design Principles for Chloroplast Genome Engineering:

• The natural repositioning of atpH in R. austro-oreganus and R. occidentalis demonstrates the plasticity of chloroplast genomes and identifies permissible rearrangement sites

• This natural experiment reveals that essential genes like atpH can function in alternative genomic contexts, providing confidence for synthetic reorganization of chloroplast genomes

• The expanded SSC region (~21.2 kb) in these species identifies potential "buffer zones" that can accommodate inserted genetic elements without disrupting essential functions

2. Optimizing Gene Expression Through Strategic Positioning:

• The different genomic contexts of atpH (LSC vs. SSC regions) provide a natural case study in how positional effects influence gene expression

• Synthetic biologists could leverage this knowledge to strategically position transgenes in chloroplast genomes for optimal expression levels

• Comparing expression efficiency of atpH in its different natural positions could inform design rules for synthetic chloroplast circuits

3. Engineering Stress-Responsive ATP Synthase Systems:

• The evolutionary selection pressure that likely drove atpH rearrangement may be related to environmental adaptation

• Analysis of whether the repositioned atpH correlates with altered ATP synthase performance under specific environmental conditions could inspire stress-responsive synthetic systems

• Comparative functional analysis between standard and rearranged atpH contexts might reveal optimizations for different growth conditions

4. Synthetic Minimal Chloroplast Genomes:

• The functional viability of species with rearranged atpH demonstrates the robustness of chloroplast genetic systems

• This supports the feasibility of designing minimal chloroplast genomes with optimized gene arrangements

• Natural rearrangements provide evidence for which gene adjacencies are non-essential and can be modified in synthetic designs

5. Evolutionary Computing Approaches:

• The natural experiment of atpH repositioning provides training data for algorithms that predict viable chloroplast genome arrangements

• Machine learning models could be developed to predict the functional consequences of synthetic rearrangements based on natural examples

• In silico evolution experiments could explore the adaptive landscape of potential chloroplast genome organizations

By studying these natural genomic innovations in Ranunculus species, synthetic biologists gain valuable insights into the design principles governing chloroplast genome organization and function, potentially leading to improved synthetic photosynthetic systems with enhanced efficiency or stress tolerance.