Recombinant Myxococcus xanthus ATP synthase subunit b (atpF)

What is the genomic organization of ATP synthase genes in Myxococcus xanthus?

ATP synthase genes in M. xanthus are organized in an operon structure similar to other bacteria. While specific details of the atpF gene are not directly addressed in the available literature, we can draw parallels from other M. xanthus operons. For instance, the rfbABC operon, which encodes an ATP-binding cassette (ABC) transporter, has been located on a 14-kb cloned fragment of the M. xanthus chromosome with a 7-kb region containing the complete locus . Similarly, ATP synthase genes typically maintain a conserved arrangement across bacterial species, with atpF encoding the b subunit that forms part of the peripheral stalk of the F1F0-ATP synthase complex.

How does ATP synthase function relate to the complex lifecycle of Myxococcus xanthus?

M. xanthus undergoes a complex developmental cycle involving aggregation and fruiting body formation under starvation conditions . Energy homeostasis, mediated by ATP synthase, is critical during these transitions. Under nutrient-rich conditions, M. xanthus cells glide on soil surfaces searching for organic matter, while starvation triggers a developmentally regulated signaling cascade that induces aggregation and the formation of multicellular fruiting bodies containing environmentally resistant spores . These developmental stages likely require precise regulation of ATP synthase activity to manage energy demands during the transition from vegetative growth to development.

What are the structural characteristics of ATP synthase subunit b in bacteria like M. xanthus?

ATP synthase subunit b typically contains a single N-terminal transmembrane helix anchored in the membrane and an extended α-helical domain that forms part of the peripheral stalk. While M. xanthus atpF structure hasn't been specifically characterized in the available literature, we can infer its properties based on other membrane proteins in this organism. For example, M. xanthus encodes several P-type ATPases with defined transmembrane domains and specific modular organizations, including copper-translocating P1B-ATPases . The study of these related transmembrane proteins provides methodological approaches applicable to atpF structural analysis.

What expression systems are optimal for recombinant M. xanthus atpF production?

Based on approaches used for other M. xanthus proteins, several expression systems can be considered:

• E. coli-based expression systems using vectors with strong promoters (T7, tac)

• Addition of purification tags (His-tag, similar to the His8-tag used for FruA DNA-binding domain)

• Codon optimization for the expression host

• Growth at reduced temperatures (16-25°C) to enhance proper folding

• Inclusion of chaperones to assist with membrane protein folding

The optimal approach often requires screening multiple constructs with variations in tag position (N- or C-terminal), linker sequences, and expression conditions to maximize yield and functionality.

What purification strategies yield the highest purity and activity for membrane proteins like atpF?

Purification of membrane proteins requires specialized approaches:

For maximum stability, the purification buffer should contain glycerol (10-15%), appropriate detergent at 2-3× CMC, and potentially lipids to maintain the native environment.

How can researchers overcome expression challenges specific to atpF?

Membrane proteins like atpF present specific challenges that can be addressed through:

• Construct design modifications:

  • Expression of the soluble domain only

  • Creation of fusion proteins with solubility-enhancing partners

  • Introduction of stabilizing mutations

• Expression conditions optimization:

  • Induction at lower OD600 values (0.4-0.6)

  • Extended expression times at reduced temperatures

  • Supplementing media with specific ions or cofactors

• Alternative expression approaches:

  • Cell-free protein synthesis in the presence of lipids or nanodiscs

  • Use of specialized E. coli strains (C41/C43) designed for membrane protein expression

  • Baculovirus expression systems for complex proteins

What methods are most effective for assessing ATP synthase activity in reconstituted systems?

ATP synthase activity can be measured using several complementary approaches:

• ATP synthesis assays:

  • Reconstitution of purified enzyme into proteoliposomes

  • Generation of proton gradient (pH jump or K+ diffusion with valinomycin)

  • Quantification of ATP production using luciferase-based detection

• ATP hydrolysis assays:

  • Spectrophotometric monitoring of inorganic phosphate release

  • Coupled enzyme assays tracking NADH oxidation

  • Inhibitor studies using oligomycin or DCCD

• Proton pumping measurements:

  • Fluorescent pH indicators to monitor lumen acidification

  • Potential-sensitive dyes to measure membrane potential

  • Stopped-flow kinetic analysis of proton translocation rates

How can researchers distinguish between proper assembly and misfolded recombinant atpF?

Multiple biophysical and biochemical techniques can assess proper folding and assembly:

• Structural integrity assessment:

  • Circular dichroism to verify secondary structure content

  • Thermal stability measurements via differential scanning fluorimetry

  • Limited proteolysis to probe for exposed cleavage sites

• Complex formation verification:

  • Blue native PAGE to visualize intact ATP synthase complexes

  • Co-immunoprecipitation with antibodies against other subunits

  • Analytical ultracentrifugation to determine complex size

• Functional validation:

  • ATP synthesis/hydrolysis coupling ratio determination

  • Proton translocation efficiency measurements

  • Inhibitor binding studies

What approaches can be used to study protein-protein interactions involving atpF?

Several techniques can characterize interactions between atpF and other ATP synthase components:

• Yeast two-hybrid system:

  • Similar to approaches used to study other M. xanthus proteins like FrzZ

  • Allows screening for binary protein-protein interactions

  • Requires careful construct design for membrane proteins

• Crosslinking studies:

  • Chemical crosslinkers of varying arm lengths

  • Photo-activated crosslinking for spatial precision

  • Mass spectrometry analysis of crosslinked products

• Biophysical interaction measurements:

  • Surface plasmon resonance for binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

  • Microscale thermophoresis for interactions in solution

• In vivo approaches:

  • FRET analysis between fluorescently labeled subunits

  • Split-GFP complementation assays

  • Chromatin immunoprecipitation (ChIP) techniques adapted from protocols developed for M. xanthus

What structural methods are most suitable for analyzing M. xanthus atpF?

The structural characterization of membrane proteins like atpF requires specialized approaches:

• X-ray crystallography:

  • Requires highly pure, homogeneous protein preparations

  • May benefit from antibody fragment co-crystallization

  • Often requires extensive crystallization condition screening

• Cryo-electron microscopy:

  • Increasingly powerful for membrane protein complexes

  • Can visualize ATP synthase in different conformational states

  • May reveal interaction details between b subunit and other components

• Nuclear magnetic resonance (for domains):

  • Solution NMR for soluble domains

  • Solid-state NMR for membrane-embedded regions

  • Allows dynamic studies of protein behavior

• Computational approaches:

  • Homology modeling based on related structures

  • Molecular dynamics simulations to predict behavior

  • Integrative modeling combining low and high-resolution data

How can researchers map critical functional regions within the atpF protein?

Functional mapping approaches include:

• Systematic mutagenesis:

  • Alanine scanning of conserved residues

  • Cysteine scanning for accessibility studies

  • Charge reversal mutations at predicted interaction sites

• Domain deletion and swapping:

  • Truncation analysis to identify minimal functional units

  • Creation of chimeric proteins with homologs from other species

  • Insertion of probe sequences at permissive sites

• Evolutionary analysis:

  • Identification of coevolving residue networks

  • Conservation mapping onto structural models

  • Correlation of sequence features with functional properties

What computational tools best predict structure-function relationships in atpF?

Computational approaches provide valuable insights into atpF structure and function:

These computational approaches should be validated experimentally, using techniques like those applied to study other M. xanthus proteins such as FruA DNA binding .

How is ATP synthase expression regulated during M. xanthus development?

Based on regulatory patterns observed for other M. xanthus genes, ATP synthase regulation likely involves:

• Transcriptional control mechanisms:

  • Creation of atpF-lacZ fusions to monitor promoter activity during development, similar to approaches used for copper-translocating ATPases

  • Analysis of potential developmental promoters activated during starvation

  • Identification of transcription factors binding to regulatory elements

• Signal transduction pathways:

  • Investigation of developmental signaling cascades affecting ATP synthase expression

  • Potential involvement of C-signal dependent pathways similar to those regulating Ω4400

  • Chromatin immunoprecipitation (ChIP) to identify regulatory protein binding in vivo

• Post-translational modifications:

  • Phosphorylation state changes during development

  • Potential proteolytic processing during differentiation

  • Altered subunit associations in response to environmental cues

What effect does copper stress have on ATP synthase expression and function?

The search results indicate that M. xanthus responds to copper fluctuations through regulation of copper-translocating P1B-ATPases . Similar studies for ATP synthase might reveal:

• Expression patterns under copper stress:

  • Construction of atpF-lacZ fusions to monitor expression in response to copper, similar to experiments with copA and copB genes

  • Quantitative PCR to measure transcript levels under varying copper concentrations

  • Proteomic analysis to detect changes in ATP synthase subunit abundance

• Functional impacts:

  • ATP synthesis efficiency measurements under copper stress

  • Analysis of proton leakage and coupling efficiency

  • Assessment of ATP synthase assembly and stability

• Regulatory mechanisms:

  • Identification of copper-responsive transcription factors affecting ATP synthase genes

  • Analysis of potential copper-binding motifs in ATP synthase subunits

  • Investigation of copper-dependent post-translational modifications

How do nutrient limitations affect ATP synthase composition and function during M. xanthus development?

M. xanthus undergoes dramatic developmental changes in response to starvation , which likely affect ATP synthase:

• ATP synthase remodeling during development:

  • Quantitative proteomics to track changes in subunit stoichiometry

  • Analysis of potential development-specific isoforms or modifications

  • Investigation of ATP synthase localization during fruiting body formation

• Functional adaptations:

  • Measurements of ATP synthesis rates during developmental progression

  • Analysis of proton motive force maintenance during starvation

  • Changes in regulatory properties and inhibitor sensitivity

• Energetic considerations:

  • ATP/ADP ratio monitoring during development

  • Assessment of energy charge maintenance during sporulation

  • Investigation of metabolic rewiring to support ATP synthase function

How does M. xanthus atpF compare to homologs in other bacterial species?

Comparative analysis provides evolutionary context:

• Sequence conservation patterns:

  • Multiple sequence alignment across diverse bacterial species

  • Identification of M. xanthus-specific insertions or deletions

  • Correlation of sequence features with lifestyle adaptations

• Functional comparisons:

  • Heterologous complementation studies in other bacterial systems

  • Cross-species ATP synthase subunit compatibility

  • Biochemical property differences between homologs

• Structural correlations:

  • Mapping of sequence conservation onto structural models

  • Identification of species-specific structural adaptations

  • Analysis of coevolutionary networks across species

What insights does M. xanthus atpF provide about ATP synthase evolution?

Evolutionary analysis of atpF can reveal:

• Phylogenetic relationships:

  • Construction of robust phylogenetic trees for the b subunit

  • Analysis of evolutionary rates across different domains

  • Identification of selection pressures on specific residues

• Ancestral sequence reconstruction:

  • Prediction of ancestral b subunit sequences

  • Expression and characterization of reconstructed proteins

  • Comparison with extant b subunit properties

• Structural evolution:

  • Analysis of insertion/deletion patterns across lineages

  • Investigation of domain acquisition or specialization

  • Correlation of structural features with organism complexity

How suitable is M. xanthus ATP synthase as a model for understanding ATP synthase function in other systems?

M. xanthus offers several advantages as a model system:

• Benefits as a model organism:

  • Well-established genetic manipulation methodologies

  • Complex multicellular behaviors providing physiological context

  • Availability of multiple analytical techniques established for M. xanthus

• Translational potential:

  • Insights into bacterial energy homeostasis during development

  • Understanding of ATP synthase regulation under stress conditions

  • Applications to biotechnology and synthetic biology

• Limitations and considerations:

  • Species-specific adaptations that may not be generalizable

  • Specialized developmental program not present in most bacteria

  • Technical challenges of working with a non-model organism

How can engineered variants of M. xanthus atpF contribute to bioenergetic studies?

Protein engineering approaches enable novel applications:

• Structure-function investigations:

  • Introduction of spectroscopic probes at strategic positions

  • Creation of disulfide crosslinks to restrict conformational changes

  • Site-directed spin labeling for electron paramagnetic resonance studies

• Mechanistic analyses:

  • Design of chimeric constructs to identify critical functional elements

  • Introduction of mutations affecting coupling efficiency

  • Engineering of variants with altered ion specificities

• Biophysical applications:

  • Development of FRET-based sensors for conformational changes

  • Creation of light-activated ATP synthase variants

  • Design of atpF constructs with altered mechanical properties

What approaches enable studying the dynamics of ATP synthase rotation in M. xanthus?

Single-molecule techniques provide insights into ATP synthase mechanics:

• Fluorescence-based approaches:

  • Single-molecule FRET to detect conformational changes

  • Fluorescent bead attachment to visualize rotation

  • High-speed total internal reflection fluorescence microscopy

• Force measurement techniques:

  • Magnetic tweezers to apply controlled torque

  • Atomic force microscopy to probe mechanical properties

  • Optical trapping to measure force generation

• Experimental considerations:

  • Design of immobilization strategies

  • Optimization of reconstitution into supported membranes

  • Development of real-time ATP synthesis measurement methods

How can recombinant M. xanthus ATP synthase be utilized in synthetic biology applications?

ATP synthase has potential applications in synthetic systems:

• Energy harvesting devices:

  • Integration into artificial cell systems

  • Creation of ATP-generating bioreactors

  • Development of biomimetic energy conversion devices

• Biosensor applications:

  • Design of ATP synthase-based proton gradient sensors

  • Development of inhibitor screening platforms

  • Creation of environmental contaminant detection systems

• Therapeutic relevance:

  • Screening platforms for antimicrobial compounds

  • Understanding of energy homeostasis in biofilm formation

  • Insights into bacterial persistence mechanisms