Recombinant Acholeplasma laidlawii ATP synthase subunit c (atpE)

What is Acholeplasma laidlawii ATP synthase subunit c and why is it significant?

Acholeplasma laidlawii ATP synthase subunit c (atpE) is a critical component of the F-type ATP synthase complex found in this mycoplasma species. It functions as part of the F0 sector and plays an essential role in the proton pumping process that drives ATP synthesis. Its significance stems from A. laidlawii's unique status as the only mycoplasma capable of surviving outside a host organism, making it a valuable model for studying membrane protein function and energy metabolism in minimal cellular systems . The protein is also known by alternative names including ATP synthase F0 sector subunit c, F-type ATPase subunit c, F-ATPase subunit c, and lipid-binding protein .

How should recombinant A. laidlawii ATP synthase subunit c be stored and handled in a laboratory setting?

For optimal research outcomes, recombinant A. laidlawii ATP synthase subunit c should be stored in a Tris-based buffer with 50% glycerol at -20°C, or at -80°C for extended storage periods. Working aliquots can be maintained at 4°C for up to one week . Critical considerations include:

• Avoiding repeated freeze-thaw cycles as this can significantly degrade protein integrity and functionality

• Storing small working aliquots rather than repeatedly accessing the stock solution

• Maintaining proper temperature control during experimental procedures

• Using appropriate protease inhibitors during extraction and purification processes to preserve protein structure

How does A. laidlawii ATP synthase subunit c compare functionally to its counterparts in other organisms?

A. laidlawii ATP synthase subunit c shares functional homology with other F-type ATP synthases but has evolved specific adaptations. In most organisms, ATP synthase subunit c assembles into a cylindrical c-ring oligomer that works with subunit a in the proton pumping process essential for ATP synthesis .

What post-translational modifications have been identified in A. laidlawii ATP synthase subunit c and how might they affect function?

Proteomic analyses of Acholeplasma laidlawii have revealed that numerous proteins in this organism undergo post-translational modifications (PTMs), primarily phosphorylation and acylation . While the specific PTM status of ATP synthase subunit c in A. laidlawii is not explicitly detailed in the available data, the presence of these modifications in membrane-associated proteins is significant.

In the broader proteome, 74 candidate phosphorylated proteins were identified, with 11 being integral membrane proteins, suggesting active membrane-associated signaling pathways . Additionally, 20 acylated proteins were detected, with 14 containing palmitic chains and 6 containing stearic chains . These modifications are particularly relevant for membrane proteins like ATP synthase subunit c, as they can:

• Alter protein-lipid interactions in the membrane environment

• Modify oligomerization properties of the c-ring

• Impact proton conductance through the F0 sector

• Affect protein stability and turnover rates

Researchers studying A. laidlawii ATP synthase should consider these potential modifications when designing experiments, particularly when comparing recombinant protein function to native protein behavior.

What are the optimal experimental conditions for functional assays of recombinant A. laidlawii ATP synthase subunit c?

When designing functional assays for recombinant A. laidlawii ATP synthase subunit c, researchers should consider the following experimental parameters to maximize physiological relevance and data quality:

For proton translocation assays, pH-sensitive fluorescent dyes can be used to monitor ATP synthase activity. For direct ATP synthesis measurements, luciferin-luciferase assays provide sensitive detection of ATP production. When working with the isolated subunit c, reconstitution into liposomes may be necessary to assess its contribution to the complete ATP synthase complex function .

How can researchers effectively address challenges in expressing and purifying functional recombinant A. laidlawii ATP synthase subunit c?

Expression and purification of functional recombinant A. laidlawii ATP synthase subunit c presents several challenges due to its hydrophobic nature and membrane integration requirements. A methodological approach to overcome these challenges includes:

• Expression System Selection:

  • Bacterial systems like E. coli BL21(DE3) with specialized vectors containing fusion tags that enhance solubility

  • Cell-free expression systems for direct incorporation into artificial membranes

  • Consideration of codon optimization for the expression host

• Solubilization Strategy:

  • Use of mild detergents that preserve protein structure

  • Inclusion of lipids during solubilization to maintain native-like environment

  • Stepwise detergent exchange during purification to optimize stability

• Purification Protocol:

  • Immobilized metal affinity chromatography (IMAC) using histidine tags

  • Size exclusion chromatography to separate oligomeric states

  • Validation of protein folding using circular dichroism spectroscopy

• Functional Verification:

  • Reconstitution into liposomes for proton translocation assays

  • Assessment of oligomerization properties

  • Binding studies with known interaction partners

Researchers should be particularly attentive to maintaining the protein in conditions that prevent aggregation while preserving the native structure necessary for function. SDS-PAGE analysis using methods similar to those described for A. laidlawii proteins (using 7.5% T or 16.5% T and 2.6% C gels according to the Laemmli method) can be used to assess protein purity .

What techniques are most effective for studying the interaction between A. laidlawii ATP synthase subunit c and other components of the ATP synthase complex?

Studying protein-protein interactions within the ATP synthase complex requires specialized techniques that can capture transient or stable associations in a membrane environment. The most effective approaches include:

• Chemical Cross-linking coupled with Mass Spectrometry (XL-MS):

  • Creates covalent bonds between interacting proteins

  • Identifies specific residues involved in protein-protein interactions

  • Provides spatial constraints for structural modeling

• Förster Resonance Energy Transfer (FRET):

  • Allows real-time monitoring of protein interactions in native-like conditions

  • Can detect conformational changes during ATP synthase operation

  • Requires careful selection of fluorophore attachment sites

• Co-immunoprecipitation with Antibodies Against ATP Synthase Components:

  • Captures intact protein complexes from native membranes

  • Can be combined with mass spectrometry for comprehensive interaction mapping

  • Requires antibodies specific to A. laidlawii ATP synthase components

• Native Gel Electrophoresis:

  • Preserves protein-protein interactions during separation

  • Allows visualization of intact ATP synthase complexes

  • Can be followed by mass spectrometry for subunit identification

When interpreting interaction data, researchers should consider that the c-subunit functions as part of a cylindrical oligomer (c-ring) that directly interacts with subunit a in the proton pumping process. The intact ATP synthase complex includes both the membrane-embedded F0 sector (containing subunits a, b, and c) and the catalytic F1 sector .

How can researchers use recombinant A. laidlawii ATP synthase subunit c as a model for studying membrane protein folding and assembly?

Recombinant A. laidlawii ATP synthase subunit c offers an excellent model system for studying fundamental principles of membrane protein folding and assembly due to several advantageous characteristics:

• Compact Size and Structure: The 86-amino acid protein provides a manageable system for tracking folding intermediates and assembly states .

• Oligomerization Capacity: The natural tendency of subunit c to form c-ring oligomers presents opportunities to study controlled multi-subunit assembly processes in membrane environments.

• Experimental Approaches:

  • Time-resolved fluorescence spectroscopy to monitor folding kinetics

  • Hydrogen-deuterium exchange mass spectrometry to identify folding domains

  • Site-directed mutagenesis to establish structure-function relationships

  • In vitro translation systems combined with artificial membranes to observe de novo insertion

• Comparative Analysis: Differences between A. laidlawii ATP synthase subunit c and its counterparts in other organisms can provide insights into evolutionary adaptations in membrane protein structure and function .

To maximize research value, experiments should be designed to systematically vary lipid composition, pH, temperature, and ionic strength to determine how these factors influence folding pathways and oligomeric assembly.

What approaches can be used to investigate the role of A. laidlawii ATP synthase subunit c in proton translocation?

Investigating the proton translocation mechanism of A. laidlawii ATP synthase subunit c requires specialized techniques that can detect proton movement across membranes. Effective methodological approaches include:

• Reconstitution in Proteoliposomes:

  • Incorporation of purified subunit c into artificial liposomes

  • Creation of defined proton gradients across the membrane

  • Measurement of gradient dissipation rates with and without inhibitors

• Site-Directed Mutagenesis of Key Residues:

  • Identification of conserved amino acids potentially involved in proton transport

  • Systematic mutation followed by functional assays

  • Correlation of structural changes with functional effects

• Proton Transport Assays:

  • pH-sensitive fluorescent dyes (e.g., ACMA, pyranine) to monitor proton movement

  • Stopped-flow spectroscopy for kinetic analysis

  • Patch-clamp electrophysiology for direct measurement of proton currents

• Inhibitor Studies:

  • Application of known ATP synthase inhibitors (e.g., oligomycin, DCCD)

  • Development of specific inhibitors targeting A. laidlawii ATP synthase

  • Structure-activity relationship analysis of inhibitor binding

These approaches can reveal how subunit c contributes to the coupling of proton movement to ATP synthesis and how this mechanism may differ from other bacterial and mammalian systems. The functional relationship between subunit c and subunit a should be a particular focus, as these components directly cooperate in the proton pumping process .

How do lipid interactions influence the stability and function of A. laidlawii ATP synthase subunit c?

Lipid interactions are crucial for the stability and function of membrane proteins like ATP synthase subunit c. For A. laidlawii, which can adapt to various environments, these interactions are particularly significant:

• Lipid Composition Effects:

  • A. laidlawii can modify its membrane lipid composition in response to environmental conditions

  • The cylindrical c-ring structure creates a lipid-filled central cavity that may be important for function

  • Specific lipid-protein interactions may stabilize the oligomeric assembly

• Experimental Approaches to Study Lipid Interactions:

  • Lipid binding assays using fluorescent lipid analogs

  • Molecular dynamics simulations of protein-lipid systems

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

  • Mass spectrometry of intact protein-lipid complexes

• Functional Implications:

  • Lipid composition may affect proton conductance rates

  • Annular lipids may modulate conformational changes during catalysis

  • Lipid-protein interactions may influence oligomerization and complex assembly

It's worth noting that A. laidlawii's unique ability to survive in diverse environments may be partially attributed to adaptations in its membrane proteins, including ATP synthase, that allow function across varying lipid compositions . Additionally, the alternative name "lipid-binding protein" for ATP synthase subunit c highlights its intimate association with membrane lipids .

What are the potential applications of structural studies on A. laidlawii ATP synthase subunit c for drug discovery?

Structural insights into A. laidlawii ATP synthase subunit c offer promising avenues for drug discovery, particularly given the rising interest in ATP synthase as an antimicrobial target:

• Structural Determination Approaches:

  • X-ray crystallography of reconstituted c-rings

  • Cryo-electron microscopy of intact ATP synthase complexes

  • NMR studies of isolated subunit c in membrane mimetics

  • Computational modeling based on homologous structures

• Drug Discovery Applications:

  • Identification of binding pockets unique to bacterial ATP synthases

  • Structure-based virtual screening for novel inhibitors

  • Fragment-based drug design targeting the c-ring assembly

  • Development of peptidomimetics that disrupt essential protein-protein interactions

• Therapeutic Potential:

  • Although A. laidlawii itself is typically not pathogenic, structural insights may translate to related pathogenic species

  • ATP synthase inhibitors could represent a new class of antimicrobials with novel mechanisms of action

  • Understanding species-specific structural features could enable selective targeting

• Methodological Considerations:

  • High-throughput screening assays based on ATP synthesis inhibition

  • Binding affinity measurements using surface plasmon resonance or isothermal titration calorimetry

  • Cellular permeability assessments for candidate compounds

The unique adaptability of A. laidlawii to different environments may also provide insights into the flexibility and resilience of ATP synthase structures, potentially revealing targets for inhibition that compromise this adaptability .

How might comparative studies between A. laidlawii and mammalian ATP synthase subunit c inform our understanding of energy metabolism evolution?

Comparative studies between A. laidlawii and mammalian ATP synthase subunit c can provide valuable evolutionary insights:

• Evolutionary Conservation and Divergence:

  • Sequence alignment analysis to identify conserved functional domains

  • Structural comparison to elucidate adaptations to different cellular environments

  • Functional assays to determine conservation of mechanistic principles

• Isoform Specialization:

  • While mammals have three functionally non-redundant isoforms (P1, P2, P3) that differ in their mitochondrial targeting peptides, A. laidlawii has a single form

  • Investigation of how A. laidlawii achieves functional versatility with a single isoform compared to the specialized roles of mammalian isoforms

  • Analysis of regulatory mechanisms that may compensate for the lack of isoform diversity

• Adaptation to Environmental Conditions:

  • Studies on how A. laidlawii ATP synthase functions across varying temperatures, pH, and lipid compositions

  • Comparison with the more constrained operating parameters of mammalian ATP synthase

  • Investigation of structural features that confer environmental resilience

• Research Approaches:

  • Phylogenetic analysis of ATP synthase components across species

  • Creation of chimeric proteins combining domains from different species

  • Functional complementation studies in heterologous expression systems

These comparative studies may reveal fundamental principles of energy metabolism that have been conserved throughout evolution, as well as specialized adaptations that enable organisms to thrive in specific niches .

What are the best practices for validating the functionality of recombinant A. laidlawii ATP synthase subunit c in experimental systems?

Ensuring that recombinant A. laidlawii ATP synthase subunit c retains its native functionality requires a multi-faceted validation approach:

• Structural Validation:

  • Circular dichroism spectroscopy to confirm secondary structure elements

  • Size-exclusion chromatography to verify oligomeric state

  • Limited proteolysis to assess proper folding based on protease accessibility patterns

• Functional Assays:

  • ATP synthesis measurements in reconstituted systems

  • Proton translocation assays using pH-sensitive dyes

  • Binding assays with known interaction partners (e.g., other ATP synthase subunits)

• Comparative Analysis with Native Protein:

  • Side-by-side functional comparisons with protein isolated from A. laidlawii

  • Mass spectrometry to identify any missing post-translational modifications

  • Thermal stability assessments to compare conformational resilience

• Integration into Larger Complexes:

  • Assembly assays with other ATP synthase components

  • Electron microscopy to visualize complex formation

  • Activity measurements of reconstituted complexes

Researchers should establish clear acceptance criteria for each validation method and consider how the chosen expression system, purification strategy, and experimental conditions might affect protein functionality .

How can researchers effectively use isotope labeling of A. laidlawii ATP synthase subunit c for structural and dynamic studies?

Isotope labeling provides powerful tools for investigating protein structure, dynamics, and interactions at molecular resolution:

• Types of Isotope Labeling for A. laidlawii ATP synthase subunit c:

  • Uniform ¹⁵N and/or ¹³C labeling for NMR spectroscopy

  • Selective amino acid labeling for specific structural questions

  • Deuteration to improve NMR resolution for larger assemblies

  • Site-specific incorporation of unnatural amino acids with specialized probes

• Expression Systems for Isotope Labeling:

  • E. coli grown in minimal media with isotope-enriched nitrogen and carbon sources

  • Cell-free protein synthesis systems with controlled isotope incorporation

  • Segmental labeling for focused analysis of specific protein regions

• Analytical Applications:

  • Solution and solid-state NMR to determine structure and dynamics

  • Hydrogen-deuterium exchange mass spectrometry to probe solvent accessibility

  • Neutron scattering to distinguish protein-lipid interactions

  • FTIR spectroscopy with ¹³C labeling for secondary structure analysis

• Dynamic Studies:

  • Relaxation dispersion NMR to identify conformational exchange processes

  • Time-resolved studies of assembly and functional cycles

  • Investigation of proton transfer pathways using pH-dependent NMR

For membrane proteins like ATP synthase subunit c, detergent selection is critical when using isotope labeling, as some detergents can interfere with NMR measurements. Alternative membrane mimetics such as nanodiscs or amphipols may provide better environments for structural studies .

What considerations are important when designing mutagenesis studies of A. laidlawii ATP synthase subunit c?

Mutagenesis studies of A. laidlawii ATP synthase subunit c require careful planning to yield meaningful insights into structure-function relationships:

• Target Selection Strategies:

  • Evolutionary conservation analysis to identify functionally important residues

  • Homology modeling to predict structurally critical positions

  • Literature review of equivalent positions in homologous proteins

  • Consideration of transmembrane topology and lipid-facing residues

• Types of Mutations to Consider:

  • Conservative substitutions to probe subtle functional effects

  • Charge alterations to investigate electrostatic contributions

  • Cysteine substitutions for accessibility studies and cross-linking

  • Alanine scanning to identify essential side chain contributions

• Experimental Design Considerations:

  • Expression system selection based on mutation type and desired analysis

  • Control experiments with wild-type protein processed identically

  • Dose-response measurements to quantify partial functional effects

  • Combination mutations to investigate cooperative interactions

• Analytical Framework:

  • Systematic organization of mutation results in structure-function maps

  • Statistical analysis to distinguish significant effects from experimental variation

  • Integration with computational predictions for mechanism refinement

  • Consideration of both direct and allosteric effects of mutations

A particularly valuable approach is to correlate mutagenesis results with the unique ability of A. laidlawii to adapt to various environments, potentially revealing residues that contribute to this versatility .