Recombinant Photobacterium profundum ATP synthase subunit alpha 2 (atpA2), partial

What is Photobacterium profundum ATP synthase and what distinguishes its alpha 2 subunit?

Photobacterium profundum ATP synthase is a membrane-bound enzyme complex found in the deep-sea bacterium Photobacterium profundum (strain SS9), which is adapted to high-pressure environments. Similar to other bacterial ATP synthases, it catalyzes the synthesis of ATP from ADP and inorganic phosphate using energy from transmembrane proton motive force . The alpha subunit (atpA2) is one of the major components of the F1 catalytic portion of the ATP synthase complex and contains nucleotide binding sites. While the gamma chain (ATPG2) plays a crucial role in the rotary mechanism of the enzyme , the alpha subunit forms part of the stationary hexameric ring (α3β3) that houses the catalytic sites at the interface between alpha and beta subunits . The partial recombinant version of this protein is specifically used for investigating structure-function relationships in pressure-adapted ATP synthases.

How does the ATP synthase from Photobacterium profundum compare structurally with other bacterial ATP synthases?

The ATP synthase from Photobacterium profundum shares the core architectural features seen in other bacterial ATP synthases, with a simpler subunit composition compared to mitochondrial counterparts. The bacterial F1 region typically consists of subunits α3β3γδε, while the FO region usually comprises subunits ab2c9-15 . Specifically for Photobacterium profundum:

Unlike thermophilic ATP synthases (such as from Bacillus PS3) that show adaptation to high temperatures through increased ionic interactions , P. profundum ATP synthase likely contains adaptations to high pressure environments, though these specific structural adaptations remain to be fully characterized through detailed comparison studies.

What expression systems are typically used for producing recombinant Photobacterium profundum ATP synthase subunits?

Recombinant Photobacterium profundum ATP synthase subunits are typically expressed using E. coli expression systems, similar to what is used for the gamma chain (ATPG2) . The methodology involves:

• Cloning the gene encoding the desired subunit (e.g., atpA2) into an appropriate expression vector

• Transformation into a suitable E. coli strain optimized for protein expression

• Induction of protein expression under controlled conditions

• Purification using affinity chromatography via attached tags

Most commonly, the proteins are produced with affinity tags such as N-terminal polyhistidine (His) tags or C-terminal tags like Myc to facilitate purification . For the alpha subunit specifically, expression parameters similar to those established for the gamma subunit would be applied, with optimization for the particular characteristics of the alpha subunit protein.

What are the key considerations for optimizing functional studies of recombinant P. profundum ATP synthase alpha subunit?

Conducting functional studies with the recombinant P. profundum ATP synthase alpha subunit requires careful consideration of several critical factors:

• Protein Solubility and Stability Management:

  • Buffer optimization is essential, with Tris/PBS-based buffers (pH 8.0) typically providing good results

  • Adding 5-50% glycerol improves stability during storage and handling

  • For lyophilized preparations, inclusion of 6% trehalose helps maintain protein structure during freeze-drying and reconstitution

• Reconstitution Parameters for Activity Preservation:

  • Careful reconstitution in deionized sterile water to 0.1-1.0 mg/mL

  • Addition of 5-50% glycerol (final concentration) for stability

  • Gentle handling to prevent denaturation while ensuring homogeneity

• Pressure Adaptation Considerations:

  • Studies should account for the pressure adaptation of P. profundum

  • Experimental designs may need to include high-pressure chambers to observe native functionality

  • Comparison with mesophilic bacterial ATP synthases should be conducted under both standard and high-pressure conditions

• Assembly with Other Subunits:

  • For studies requiring complete F1 assembly, all five subunits (α, β, γ, δ, and ε) must be expressed and assembled

  • Specific molar ratios of subunits should be maintained (3:3:1:1:1 for α:β:γ:δ:ε)

  • The correct assembly can be verified through size-exclusion chromatography and negative-stain electron microscopy

How can researchers investigate the rotational states and catalytic mechanism of P. profundum ATP synthase using the recombinant alpha subunit?

Investigating rotational states and catalytic mechanisms of P. profundum ATP synthase requires sophisticated experimental approaches:

• Cryo-EM Analysis Protocol:

  • Expression of complete ATP synthase complex or reconstitution from individual subunits including the recombinant alpha subunit

  • Purification through affinity chromatography followed by size exclusion chromatography

  • Sample preparation for cryo-EM with optimization of ice thickness and particle distribution

  • Data collection with motion correction and CTF estimation

  • Particle picking and classification to identify different rotational states

  • Focused refinement of specific regions (F1 and FO) to improve resolution

• Rotational State Analysis:

  • The ATP synthase typically exhibits three main rotational states due to the symmetry mismatch between the 120° steps of the F1 motor and the smaller steps of the FO motor

  • Comparison of the positions of c-subunits in different rotational states can reveal the step sizes (typically 3, 4, and 3 c-subunits per 120° F1 rotation)

  • The alpha subunit's conformational changes during these rotational states provide insight into the catalytic mechanism

• Site-Directed Mutagenesis Studies:

  • Targeted mutations in key residues of the alpha subunit can identify essential amino acids for catalysis

  • Comparison of equivalent mutations in mesophilic ATP synthases can highlight pressure-specific adaptations

  • Activity assays under various pressure conditions can correlate structural features with functional adaptations

What methods can be used to study the effect of high pressure on the structure and function of P. profundum ATP synthase alpha subunit?

Studying pressure effects on P. profundum ATP synthase alpha subunit requires specialized methodologies:

• High-Pressure Biophysical Techniques:

  • High-pressure X-ray crystallography to determine structural changes under pressure

  • High-pressure NMR spectroscopy to analyze dynamic changes in protein conformation

  • High-pressure circular dichroism to monitor secondary structure stability

  • Pressure perturbation calorimetry to measure volumetric properties and hydration changes

• Functional Assays Under Pressure:

  • High-pressure stopped-flow spectroscopy to measure kinetic parameters

  • Enzyme activity assays in pressure chambers to determine pressure optima and ranges

  • Comparison of ATP synthesis/hydrolysis rates at different pressures

  • Measurement of proton translocation efficiency under pressure conditions

• Comparative Analysis Framework:

  Parameter	Atmospheric Pressure	Moderate Pressure (50 MPa)	High Pressure (100 MPa)
  ATP Synthesis Rate	Baseline measurement	Expected increase for P. profundum	Optimal activity expected
  Structural Stability	Baseline stability	Enhanced stability for pressure-adapted features	Maintained functionality
  Conformational Flexibility	Standard measurement	Potentially reduced	Optimized for function
  Subunit Interaction Strength	Standard measurement	Potentially enhanced	Optimized for function

• Molecular Dynamics Simulations:

  • Simulation of alpha subunit behavior under different pressure conditions

  • Analysis of water molecule organization around the protein at high pressure

  • Identification of pressure-sensing regions within the protein structure

  • Prediction of pressure-induced conformational changes that can be verified experimentally

How should researchers design experiments to compare P. profundum ATP synthase alpha subunit with homologs from non-piezophilic bacteria?

Designing comparative experiments between P. profundum ATP synthase alpha subunit and non-piezophilic homologs requires:

• Homolog Selection Strategy:

  • Choose homologs from phylogenetically related bacteria living at different depths/pressures

  • Include both mesophilic (e.g., E. coli) and other extremophile (thermophilic, psychrophilic) homologs

  • Ensure sequence alignment and homology modeling to identify key structural differences

• Standardized Expression and Purification Protocol:

  • Express all homologs using identical expression systems and conditions

  • Use the same affinity tags (N-terminal His tag or C-terminal Myc tag) for all proteins

  • Implement identical purification protocols to eliminate methodology-based variations

  • Verify protein purity (>90%) through SDS-PAGE analysis

• Systematic Comparative Assays:

  • Thermal stability assays (differential scanning fluorimetry) at various pressures

  • Nucleotide binding affinity measurements under pressure gradients

  • Structural analysis through circular dichroism at different pressures

  • Limited proteolysis experiments to identify domains with different pressure sensitivities

• Data Analysis Framework:

  Analysis Parameter	Measurement Technique	Expected Differences
  Pressure stability	Half-life at elevated pressure	Longer for P. profundum
  Conformational changes	Intrinsic fluorescence spectroscopy	Smaller changes for P. profundum at high pressure
  Activity profile vs. pressure	ATP synthesis assay	Broader optimum for P. profundum
  Volume change upon nucleotide binding	Pressure perturbation calorimetry	Smaller for P. profundum

What are the key considerations for designing site-directed mutagenesis studies of P. profundum ATP synthase alpha subunit?

Site-directed mutagenesis studies of P. profundum ATP synthase alpha subunit should consider:

• Target Residue Identification Strategy:

  • Analyze sequence alignments with non-piezophilic homologs to identify unique residues

  • Focus on ionizable residues that may contribute to pressure adaptation

  • Target residues in nucleotide binding domains and subunit interfaces

  • Examine regions with potential flexibility differences under pressure

• Mutation Design Principles:

  • Create conservative mutations (similar physicochemical properties) first

  • Design reverse mutations (changing P. profundum-specific residues to those found in mesophilic homologs)

  • Create charge-altering mutations to test electrostatic interaction hypotheses

  • Design volume-changing mutations to test packing hypotheses for pressure adaptation

• Experimental Validation Hierarchy:

  • Expression level and solubility screening

  • Structural integrity verification through circular dichroism

  • Thermal and pressure stability profiling

  • Nucleotide binding affinity determination

  • Assembly capacity with other ATP synthase subunits

  • Functional testing through ATP synthesis/hydrolysis assays under pressure

• Integrative Data Analysis Approach:

  • Correlation of structural changes with functional outcomes

  • Multi-parameter analysis to identify synergistic effects of mutations

  • Development of a molecular model for pressure adaptation mechanisms

  • Statistical analysis to determine significance of observed differences

How can researchers reconstitute functional ATP synthase complexes incorporating the recombinant P. profundum alpha subunit for structural studies?

Reconstituting functional ATP synthase complexes with recombinant P. profundum alpha subunit requires:

• Component Preparation Protocol:

  • Expression and purification of all necessary subunits (α, β, γ, δ, ε, a, b, c) with appropriate tags

  • Verification of purity (>90%) for each subunit through SDS-PAGE

  • Solubilization of membrane subunits (a, b, c) in appropriate detergents

  • Removal of tags if they interfere with assembly or function

• Reconstitution Strategy:

  • Sequential addition of subunits in defined order to promote correct assembly

  • Incubation under controlled conditions (temperature, pH, ionic strength)

  • Verification of assembly through size-exclusion chromatography

  • Functional verification through ATP synthesis/hydrolysis assays

• Structural Analysis Methods:

  • Negative-stain electron microscopy for initial structural verification

  • Cryo-EM sample preparation optimization

  • Data collection with high-end electron microscopes

  • Image processing following established protocols for ATP synthases

  • Focused refinement of the F1 and FO regions to improve resolution

• Sample Preparation for Different Structural Methods:

  Method	Sample Requirements	Special Considerations
  Cryo-EM	2-5 mg/ml protein, minimal detergent	Grid optimization, preventing preferential orientation
  X-ray Crystallography	10-20 mg/ml protein, highly pure	Crystallization condition screening, pressure chambers
  Solution NMR	Isotopically labeled proteins	Size limitations, subunit interaction focus
  Solid-state NMR	Reconstituted in lipid environment	Membrane mimetic optimization
  SAXS/SANS	1-5 mg/ml protein	Contrast matching for subunit localization

What are common challenges in expressing and purifying recombinant P. profundum ATP synthase subunits and how can they be addressed?

Researchers commonly encounter several challenges when working with recombinant P. profundum ATP synthase subunits:

• Expression Yield Optimization:

  • Challenge: Low expression levels due to codon bias in E. coli

  • Solution: Use codon-optimized gene sequences and specialized E. coli strains with rare codons (e.g., Rosetta)

  • Challenge: Protein misfolding due to rapid expression

  • Solution: Lower induction temperature (16-20°C) and reduce inducer concentration

• Solubility Enhancement Strategies:

  • Challenge: Formation of inclusion bodies

  • Solution: Co-express with chaperones, use solubility tags (MBP, SUMO), or optimize lysis buffer composition

  • Challenge: Aggregation during purification

  • Solution: Include glycerol (5-50%) in all buffers and maintain protein at 4°C throughout purification

• Purification Troubleshooting:

  • Challenge: Low affinity to Ni-NTA resin despite His-tag

  • Solution: Ensure tag is accessible, adjust imidazole concentration in binding buffer, try different metal ions

  • Challenge: Co-purification of contaminants

  • Solution: Increase washing stringency, add secondary purification steps (ion exchange, size exclusion)

• Stability During Storage:

  • Challenge: Activity loss during freeze-thaw cycles

  • Solution: Aliquot protein and store at -80°C, include cryoprotectants like glycerol (50% final)

  • Challenge: Precipitation during storage

  • Solution: Consider lyophilization with 6% trehalose as a stabilizer

How should researchers interpret inconsistencies between structural data and functional assays of P. profundum ATP synthase?

When faced with discrepancies between structural data and functional assays, researchers should:

• Systematic Discrepancy Analysis:

  • Evaluate whether the recombinant protein truly represents the native state

  • Consider if the experimental conditions (especially pressure) match physiological conditions

  • Assess whether tags or modifications affect function or structure

  • Determine if the protein is in the expected rotational/conformational state for observed function

• Structure-Function Correlation Framework:

  • Map functional data onto structural models to identify correlations and discrepancies

  • Consider that different rotational states may explain functional variances

  • Analyze whether observed inconsistencies relate to known flexible regions

  • Determine if subunit interactions in reconstituted systems match native complexes

• Methodological Validation Approach:

  • Verify structural data through orthogonal structural methods

  • Confirm functional data using multiple assay types and conditions

  • Test function under conditions that match structural studies (buffer, temperature, pressure)

  • Consider time-resolved approaches to capture dynamic states missed in static structural studies

• Biological Context Interpretation:

  • Compare with data from related organisms to distinguish organism-specific features

  • Consider evolutionary context and pressure adaptation mechanisms

  • Assess whether discrepancies might represent actual regulatory mechanisms

  • Evaluate if the differences reveal novel aspects of ATP synthase function under pressure

What analytical techniques are most effective for studying the proton translocation pathway in P. profundum ATP synthase and interpreting the resulting data?

Studying proton translocation in P. profundum ATP synthase requires specialized techniques:

• High-Resolution Structural Analysis:

  • Cryo-EM with focused refinement of the membrane region to visualize the proton channel

  • X-ray crystallography of the FO portion (challenging but potentially high-reward)

  • Molecular dynamics simulations to model proton movement through identified channels

  • Hydrogen/deuterium exchange mass spectrometry to identify solvent-accessible regions

• Functional Proton Translocation Assays:

  • pH-sensitive fluorescent probe assays in reconstituted liposomes

  • Patch-clamp electrophysiology of reconstituted enzyme in artificial membranes

  • Measurement of proton translocation under various pressure conditions

  • Analysis of the effects of pH and membrane potential (ΔΨ) on activity, similar to studies with Bacillus PS3

• Site-Directed Mutagenesis Strategy:

  • Targeted mutations of putative proton-carrying residues

  • Creation of chimeric proteins with subunits from non-piezophilic bacteria

  • Introduction of reporter groups at key positions in the proton pathway

  • Correlation of mutation effects with structural models

• Data Integration Framework:

  Analysis Approach	Data Types Combined	Expected Insights
  Structure-Function Mapping	Cryo-EM + mutagenesis results	Identification of essential residues in proton path
  Pressure-Response Profile	Activity assays + H/D exchange	Pressure effects on channel accessibility
  Comparative Pathway Analysis	P. profundum data vs. mesophilic bacteria	Pressure-specific adaptations in proton translocation
  Integrative Modeling	All experimental data + MD simulations	Complete model of proton movement through the enzyme

How might understanding P. profundum ATP synthase contribute to designing pressure-resistant enzymes for biotechnological applications?

The study of P. profundum ATP synthase offers valuable insights for enzyme engineering:

• Pressure Adaptation Principles:

  • Identification of specific amino acid substitutions that confer pressure resistance

  • Understanding of protein packing principles that maintain function under pressure

  • Elucidation of flexibility/rigidity balance that optimizes activity at high pressure

  • Discovery of interaction networks that stabilize protein complexes under pressure

• Biomimetic Design Strategy:

  • Transfer of identified pressure-resistant motifs to industrial enzymes

  • Creation of chimeric proteins incorporating P. profundum ATP synthase domains

  • Development of computational algorithms to predict pressure-stabilizing mutations

  • Implementation of machine learning approaches to identify non-obvious pressure adaptation patterns

• Potential Biotechnological Applications:

  • Design of pressure-resistant biocatalysts for high-pressure industrial processes

  • Development of enzymes for deep-sea bioremediation

  • Creation of pressure-stable proteins for high-pressure food processing

  • Engineering of pressure-resistant biomolecular materials

• Research-to-Application Roadmap:

  Research Phase	Key Outcomes	Application Potential
  Fundamental Structure-Function Analysis	Identification of pressure adaptation motifs	Design principles for protein engineering
  Comparative Studies with Homologs	Ranking of adaptation effectiveness	Selection of best motifs for transfer
  Directed Evolution Under Pressure	Novel pressure-resistant variants	Optimization for specific applications
  Industrial Enzyme Modification	Pressure-resistant industrial enzymes	Enhanced bioprocessing under pressure

What are the most promising approaches for studying the evolutionary adaptations of ATP synthase to high-pressure environments?

Evolutionary studies of ATP synthase pressure adaptation should consider:

• Phylogenetic Analysis Framework:

  • Comprehensive sequence collection from organisms across pressure gradients

  • Construction of phylogenetic trees focusing on ATP synthase subunits

  • Identification of convergent evolution patterns in different pressure-adapted lineages

  • Correlation of sequence changes with habitat depth/pressure

• Comparative Genomics Approach:

  • Analysis of ATP synthase operon organization in piezophiles versus mesophiles

  • Identification of regulatory elements that may control expression under pressure

  • Assessment of horizontal gene transfer events that may have contributed to pressure adaptation

  • Examination of genomic context for ATP synthase genes in pressure-adapted species

• Ancestral Sequence Reconstruction:

  • Computational resurrection of ancestral ATP synthase sequences

  • Laboratory expression and characterization of ancestral proteins

  • Experimental testing of evolutionary trajectories through intermediate constructs

  • Identification of key mutations that enabled pressure adaptation

• Integration with Structural Biology:

  • Mapping of evolutionarily significant residues onto high-resolution structures

  • Correlation of evolutionary rate with structural features

  • Analysis of co-evolution patterns between interacting subunits

  • Molecular dynamics simulations of ancestral and modern proteins under pressure

How can researchers effectively combine structural biology and biophysical approaches to develop a comprehensive model of P. profundum ATP synthase function under pressure?

Developing an integrative model requires combining multiple approaches: