Recombinant Mesoplasma florum ATP synthase subunit beta (atpD)

What is the structure and function of Mesoplasma florum ATP synthase subunit beta (atpD)?

The ATP synthase subunit beta (atpD) in Mesoplasma florum is part of the F1 domain of the F1F0 ATP synthase complex. This protein produces ATP from ADP in the presence of a proton gradient across the membrane . The beta subunit contains the primary catalytic sites responsible for ATP synthesis or hydrolysis.

Based on structural homology with other bacterial ATP synthases, Mesoplasma florum atpD likely contains:

• Conserved Walker A (P-loop) motif for interaction with ATP phosphate groups

• Walker B motif involved in ATP hydrolysis

• The DELSEED-loop signature essential for interaction with regulatory subunits

The atpD protein forms part of the (αβ)3 hexameric structure in the F1 portion, which constitutes the catalytic core of the enzyme. While in most bacteria ATP synthase functions in ATP generation, in mycoplasmas including Mesoplasma florum, it may function primarily in ATP hydrolysis and maintenance of the electrochemical gradient .

How are ATP synthase components organized in the Mesoplasma florum genome?

In Mesoplasma florum, ATP synthase components are encoded by a cluster of genes in the genome. According to available data, the organization includes:

This gene organization reflects the functional components needed for the complete ATP synthase complex . Unlike other bacteria, mycoplasmas often have additional copies of atpA and atpD genes arranged in pairs outside the typical operon structure, suggesting potential specialized functions or adaptations in these minimal organisms .

What expression systems are optimal for producing recombinant Mesoplasma florum atpD?

E. coli expression systems are most commonly used for producing recombinant Mesoplasma florum proteins. E. coli codon-optimized versions of Mesoplasma florum genes, including ATP synthase components, are available in gene repositories .

For optimal expression of functional atpD, the following methodology is recommended:

• Vector selection: pET-based vectors with T7 promoter systems offer high-level expression control

• Host strain: E. coli BL21(DE3) lacking certain proteases that might degrade recombinant proteins

• Expression conditions:

  • Growth at 30°C until OD600 reaches 0.6-0.8

  • Induction with 0.5 mM IPTG

  • Post-induction cultivation at 18-25°C for 16-18 hours to enhance proper folding

Experimental evidence from studies with other bacterial ATP synthase components indicates that lower temperature expression significantly improves the yield of properly folded, functional protein .

What purification strategies are most effective for recombinant Mesoplasma florum atpD?

Purification of recombinant Mesoplasma florum atpD typically involves a multi-step chromatographic approach:

Step-by-step purification protocol:

• Cell lysis: Sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM ATP, 5 mM MgCl2, and protease inhibitors

• Clarification: Centrifugation at 20,000 × g for 30 minutes at 4°C

• Affinity chromatography:

  • For His-tagged protein: Ni-NTA affinity chromatography

  • Wash with increasing imidazole concentrations (10-40 mM)

  • Elution with 250 mM imidazole

• Tag removal: TEV protease cleavage (if applicable)

• Ion exchange chromatography: MonoQ column with a linear NaCl gradient (0-500 mM)

• Size exclusion chromatography: Superdex 200 in buffer containing 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT

• Concentration: Using 30 kDa cutoff concentrators to 5-10 mg/ml

The inclusion of ATP or ADP and Mg²⁺ in buffers is crucial for maintaining the stability and proper folding of the protein throughout the purification process .

How can researchers assess the functional activity of purified recombinant Mesoplasma florum atpD?

Several complementary methods can be used to assess the functionality of purified recombinant atpD:

• ATP hydrolysis assay:

  • Colorimetric measurement of inorganic phosphate release using malachite green

  • Typical reaction conditions: 50 mM Tris-HCl pH 8.0, 5 mM MgCl2, 2 mM ATP at 37°C

  • Expected activity for functional protein: 5-10 μmol Pi/min/mg protein

• Nucleotide binding assay:

  • Isothermal titration calorimetry (ITC) to determine binding constants

  • Typical Kd values for ATP: 10-50 μM

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to verify correct oligomeric state

• Complementation studies:

  • In vitro reconstitution with other subunits to form F1 complex

  • ATPase activity measurement of reconstituted complexes

Reference data from experimental studies with M. florum suggest that functional ATP synthase shows significant ATPase activity that can be partially inhibited by typical ATPase inhibitors like oligomycin or DCCD .

How do mutations in conserved motifs affect the ATPase activity of recombinant Mesoplasma florum atpD?

The Walker A (P-loop) and Walker B motifs are critical for nucleotide binding and hydrolysis in ATP synthase beta subunits. Based on studies in related organisms, these motifs are conserved in Mesoplasma florum atpD .

Methodologically, these studies require:

• Site-directed mutagenesis using PCR-based methods

• Expression and purification as described in basic sections

• Enzymatic activity assays measuring ATP hydrolysis

• Structural analysis using X-ray crystallography or cryo-EM to observe conformational changes

The membrane ATPase activity linked to ATP synthase subunits can be specifically assessed using methodologies similar to those described in research on mycoplasma ATP synthase, where membrane-enriched fractions are isolated and tested for ATPase activity by measuring inorganic phosphate release .

What structural features distinguish Mesoplasma florum ATP synthase from those of other bacteria?

As a near-minimal bacterium, Mesoplasma florum has likely evolved specific adaptations in its ATP synthase complex:

• Gene organization peculiarities:

  • Mycoplasmas typically contain extra copies of atpA and atpD genes arranged in pairs outside the traditional operon

  • These additional copies may have specialized functions

• Structural adaptations:

  • Possible modifications in the c-ring composition affecting proton translocation efficiency

  • Potential simplification of regulatory mechanisms in line with genome minimization

• Membrane interaction:

  • Adaptations to function in a cell wall-less organism with unique membrane properties

  • Potentially altered lipid interactions that optimize activity in Mesoplasma's specific environment

Structural studies using cryo-EM, as have been performed for ATP synthases from other organisms, would be required to fully characterize these unique properties . The transcriptome and proteome analysis methods described in research on M. florum provide a foundation for such structural studies .

How does recombinant Mesoplasma florum atpD interact with other ATP synthase subunits to form functional complexes?

The formation of functional ATP synthase complexes involves specific interactions between atpD and other subunits:

• Alpha-beta interactions:

  • The beta subunit forms heterohexamers with alpha subunits in a (αβ)3 arrangement

  • These interactions can be studied using yeast two-hybrid or pull-down assays

• Central stalk interactions:

  • The beta subunit interacts with the gamma subunit, which rotates during catalysis

  • These dynamic interactions can be studied using FRET or crosslinking experiments

• Epsilon subunit regulation:

  • The DELSEED-loop of the beta subunit interacts with the epsilon subunit (Mfl116)

  • This interaction is crucial for the inhibitory function of the epsilon subunit

To experimentally characterize these interactions:

• Blue Native PAGE can be used to analyze complex formation

• Co-immunoprecipitation with tagged versions of different subunits

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction surfaces

• In vitro reconstitution of subcomplexes to test functionality

Research on ATP synthase complexes in other organisms indicates that these protein-protein interactions are essential for both the structural integrity and functional activity of the complex .

How can recombinant Mesoplasma florum atpD be used to study the evolution of F-type ATPases in minimal genomes?

Recombinant Mesoplasma florum atpD provides a valuable tool for evolutionary studies through:

• Comparative structural analysis:

  • Alignment of Mesoplasma florum atpD with homologs from other minimal bacteria, standard bacteria, archaea, and eukaryotes

  • Identification of core conserved features versus adaptations specific to minimal genomes

  • 3D structure comparison to map evolutionary conservation onto structural features

• Functional complementation:

  • Testing whether Mesoplasma florum atpD can functionally replace the beta subunit in other species

  • Evaluation of complementation efficiency as a measure of functional conservation

• Chimeric protein studies:

  • Creating fusion proteins containing domains from Mesoplasma florum atpD and other species

  • Identifying which regions are responsible for specific functions or adaptations

• Evolutionary rate analysis:

  • Calculating dN/dS ratios to identify selective pressures on different protein domains

  • Comparing evolutionary rates between minimal bacteria and other organisms

These approaches can help understand how essential cellular machinery like ATP synthase has been maintained while genomes were minimized during evolution. The presence of extra copies of atpA and atpD genes in mycoplasmas suggests interesting evolutionary adaptations that merit further investigation .

What approaches can be used to study the membrane association of recombinant Mesoplasma florum ATP synthase components?

To study membrane association of ATP synthase components including atpD, researchers can employ several complementary approaches:

• Limited proteolysis experiments:

  • Treatment of intact cells with trypsin immobilized on agarose beads

  • Analysis of protein accessibility by western blotting with specific antibodies

  • This approach has been successfully used to determine the topology of ATP synthase components in mycoplasmas

• Membrane fractionation:

  • Isolation of membrane-enriched fractions by sonication and differential centrifugation

  • Assessment of ATPase activity in these fractions using colorimetric phosphate release assays

  • Comparison between wild-type and mutant strains to assess the contribution of specific components

• Fluorescence microscopy:

  • Expression of fluorescently tagged atpD in Mesoplasma florum

  • Visualization of localization and dynamics using high-resolution microscopy techniques

  • Co-localization studies with membrane markers

• Reconstitution in liposomes:

  • Incorporation of purified recombinant proteins into artificial liposomes

  • Measurement of ATP synthesis/hydrolysis activities

  • Assessment of proton pumping using pH-sensitive fluorescent dyes

The experimental approach described in research on mycoplasma ATP synthase, where limited proteolysis was used to determine protein topology and membrane association, provides a methodological foundation for such studies .

How can researchers generate and characterize mutant forms of Mesoplasma florum lacking functional atpD?

Creating and characterizing atpD mutants requires specific methodological approaches due to the essential nature of ATP synthase:

• Mutagenesis strategies:

  • Transposon mutagenesis to create random insertions in the genome, as demonstrated in mycoplasma research

  • CRISPR-Cas9 for targeted modifications

  • Complementation with plasmid-encoded wild-type atpD to maintain viability if the mutation is lethal

• Screening methods:

  • PCR-based screening to identify mutants with insertions or modifications in atpD

  • Phenotypic screening based on growth characteristics or resistance markers

• Characterization approaches:

  • Comparative growth studies in different media compositions

  • Measurement of ATP levels in wild-type versus mutant strains

  • Assessment of membrane potential using fluorescent dyes

  • Quantification of ATPase activity in membrane fractions

• Complementation studies:

  • Reintroduction of wild-type or modified atpD on plasmids

  • Evaluation of phenotypic rescue

  • This approach has been successfully used in mycoplasma research to confirm the role of ATP synthase components

Experimental evidence from mycoplasma research shows that mutants with disrupted ATP synthase components can be viable but show altered growth characteristics and significantly reduced ATPase activity in membrane fractions .

What techniques are most effective for studying the ATP synthase complex architecture in Mesoplasma florum?

Several advanced structural biology techniques can elucidate the complex architecture of Mesoplasma florum ATP synthase:

• Cryo-Electron Microscopy (cryo-EM):

  • High-resolution structural analysis of purified ATP synthase complexes

  • Visualization of different conformational states

  • This technique has been successfully applied to ATP synthases from other organisms, revealing unique architectural features

• Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE):

  • Separation of intact membrane protein complexes

  • Identification of different assembly states (monomers, dimers)

  • In-gel activity assays to correlate structure with function

• Crosslinking Mass Spectrometry (XL-MS):

  • Identification of protein-protein interactions within the complex

  • Mapping of spatial relationships between subunits

  • Validation of structural models

• Single-particle tracking:

  • Analysis of ATP synthase dynamics in native membranes

  • Correlation of structural states with functional activities

Research on ATP synthases from other organisms has demonstrated that these complexes can exist in different oligomeric states (monomers and dimers), with sizes of approximately 600 kDa for monomers and 1-1.2 MDa for dimers . Similar approaches could be applied to characterize the Mesoplasma florum ATP synthase complex.

How can isotope labeling techniques be applied to study ATP synthesis kinetics in Mesoplasma florum systems?

Isotope labeling provides powerful approaches to study ATP synthesis dynamics in Mesoplasma florum:

• ¹⁸O labeling for ATP synthesis/hydrolysis:

  • Use of H₂¹⁸O to track oxygen incorporation into Pi during ATP hydrolysis

  • Mass spectrometry detection of labeled products

  • Determination of reaction mechanisms and rates

• ³²P/³³P labeling for phosphate tracking:

  • Incorporation of radiolabeled phosphate into ATP

  • Time-course analysis of ATP synthesis

  • Quantification using scintillation counting or phosphorimaging

• ¹³C/¹⁵N metabolic labeling:

  • Growth of M. florum in media containing ¹³C-glucose or ¹⁵N-amino acids

  • NMR or mass spectrometry analysis of labeled ATP

  • Tracking of carbon and nitrogen flow through metabolic pathways

• Deuterium labeling for proton translocation studies:

  • Use of D₂O to study proton transfer mechanisms

  • Kinetic isotope effect analysis

  • Correlation with ATP synthesis rates

Experimental protocols would include:

• Cell growth in isotope-enriched media

• Isolation of ATP synthase components or membrane vesicles

• In vitro reconstitution of ATP synthesis systems

• Analytical detection of labeled products using mass spectrometry or NMR

These approaches can provide detailed insights into the kinetics and mechanisms of ATP synthesis in this near-minimal bacterium.

What computational approaches can inform structural and functional studies of Mesoplasma florum atpD?

Computational methods can complement experimental approaches in studying Mesoplasma florum atpD:

• Homology modeling and molecular dynamics simulations:

  • Generation of 3D structural models based on homologous proteins

  • Simulation of protein dynamics and conformational changes

  • Investigation of nucleotide binding and hydrolysis mechanisms

• Systems biology modeling:

  • Integration of atpD function into genome-scale metabolic models

  • Flux balance analysis to predict the impact of atpD modifications

  • This approach is particularly relevant for M. florum as a model for systems biology

• Evolutionary analysis:

  • Phylogenetic studies to trace the evolution of ATP synthase components

  • Analysis of selective pressure using dN/dS ratios

  • Identification of conserved residues critical for function

• Protein-protein interaction prediction:

  • Docking simulations to model interactions between atpD and other subunits

  • Prediction of binding interfaces and critical residues

  • These predictions can guide experimental mutagenesis studies

• Promoter analysis and gene expression prediction:

  • Identification of regulatory elements in the atpD promoter

  • Correlation with transcriptomic data to understand expression regulation

  • M. florum transcriptome studies have identified conserved promoter motifs that can be analyzed for ATP synthase genes

These computational approaches can guide experimental design and help interpret experimental results, ultimately leading to a more comprehensive understanding of Mesoplasma florum atpD structure, function, and regulation.