Recombinant Prochlorococcus marinus subsp. pastoris ATP synthase subunit b' (atpG)

What is the nomenclature and classification of atpG within the ATP synthase complex?

ATP synthase subunit b' from Prochlorococcus marinus is known by several alternative names in the scientific literature, which can cause confusion when reviewing research findings:

• ATP synthase F₀ sector subunit b'

• ATPase subunit II

• F-type ATPase subunit b'

• F-ATPase subunit b' (shorter name)

The protein is encoded by the atpG gene (PMM1454 in the genome annotation). Functionally, it belongs to the F-type ATP synthase family and is a component of the membrane-embedded F₀ sector. Understanding this classification is essential for comparative analyses with other ATP synthase components and for placing experimental results in the proper context of energy transduction mechanisms.

What are the optimal storage conditions for maintaining recombinant atpG stability?

For maximum stability of purified recombinant atpG protein, researchers should follow these evidence-based protocols:

• Store stock solutions at -20°C or preferably -80°C for extended storage

• Utilize storage buffers containing Tris-based components with 50% glycerol (for protein stocks) or 6% trehalose (for lyophilized preparation)

• Avoid repeated freeze-thaw cycles as this significantly reduces protein activity

• Prepare working aliquots and maintain at 4°C for up to one week

• For reconstitution of lyophilized protein, use deionized sterile water to a concentration of 0.1-1.0 mg/mL, and consider adding 5-50% glycerol for long-term storage

Adhering to these storage parameters ensures protein integrity for downstream functional and structural analyses.

What expression systems provide optimal yields of functional atpG protein?

Based on available research methodologies for similar ATP synthase components, the following expression systems have proven successful for recombinant atpG production:

The recombinant atpG protein has been successfully expressed in E. coli systems with an N-terminal His-tag, which facilitates downstream purification while maintaining protein function . For membrane proteins like atpG, the addition of chaperone proteins such as DnaK, DnaJ, and GrpE through co-transformation with plasmids like pOFXT7KJE3 can substantially increase expression yields of correctly folded protein .

What induction and extraction protocols optimize recombinant atpG recovery?

For optimal induction and extraction of recombinant atpG:

• Grow bacterial cultures to mid-log phase (OD₆₀₀ of 0.6-0.8)

• Induce expression with 1.0 mM IPTG for 30 minutes (shorter induction times may reduce toxicity effects)

• Harvest cells by centrifugation at approximately 6000 × g for 20 minutes

• Resuspend pellets in lysis buffer containing:

  • 20 mM Tris-HCl pH 8.0

  • 2% v/v Protease Inhibitor Cocktail

  • 1 mg/mL lysozyme (incubate at 4°C for 1.5 hours)

• Lyse cells by sonication at 50-75W with cooling intervals to prevent protein denaturation

• For membrane proteins like atpG, consider adding mild detergents (0.5-1% n-dodecyl β-D-maltoside) to solubilize membrane fractions

These protocols balance protein yield with the maintenance of structural integrity and function.

How can researchers verify the identity and purity of recombinant atpG?

Multiple complementary approaches should be used to confirm identity and assess purity:

• SDS-PAGE analysis: Should show >90% purity with a single band at approximately 17 kDa corresponding to the 153-amino acid atpG protein (with potential slight shift due to the His-tag)

• Western blotting: Using anti-His antibodies or specific anti-atpG antibodies; compare with native ATP synthase as a positive control if available

• Mass spectrometry verification:

  • MALDI-TOF to confirm the molecular weight

• Circular dichroism: To verify secondary structure composition and proper folding, especially important for functional studies

Researchers should aim for >90% purity as determined by densitometric analysis of SDS-PAGE gels .

How can recombinant atpG be used for structural and functional reconstitution studies?

Recombinant atpG can be employed in several advanced experimental approaches:

• Reconstitution of ATP synthase subcomplexes:

  • Combine purified atpG with other recombinant or native ATP synthase components

  • Utilize strategies similar to those developed for chloroplast ATP synthase subunit c reconstitution

  • Gradually remove detergent using bio-beads or dialysis to form proteoliposomes

• Single-molecule studies:

  • Label recombinant atpG with fluorescent probes at engineered cysteine residues

  • Monitor protein dynamics during ATP synthesis/hydrolysis

  • Evaluate interactions between atpG and other subunits using FRET techniques

• Structural analysis:

  • Incorporate recombinant atpG into 2D crystallization trials

  • Prepare samples for cryo-electron microscopy of the assembled complex

  • Utilize solution NMR for structural characterization of specific domains

The successful reconstitution of functional ATP synthase components using recombinant subunits has been demonstrated for subunit c , providing a methodological framework applicable to atpG studies.

What experimental approaches can elucidate atpG's role in Prochlorococcus adaptations?

The unique properties of Prochlorococcus marinus, including its streamlined genome and unusual regulatory features , make atpG an interesting target for understanding bioenergetic adaptations. Several experimental approaches can elucidate its role:

• Comparative analysis with related cyanobacteria:

  • Sequence alignment shows atpG from oceanic strains forms a separate subclade within cyanobacterial radiation, similar to the pattern observed with glnB

  • Functional differences may correlate with ecological niches and energy requirements

• Expression analysis under different environmental conditions:

  • RT-qPCR to quantify atpG expression under varying light intensities

  • RNA-seq to identify co-regulated genes

  • Compare with the unusual regulatory patterns observed for other genes in Prochlorococcus (e.g., glnA, amt1)

• Mutational analysis in heterologous systems:

  • Express wild-type and mutant forms in model cyanobacteria

  • Assess impact on ATP synthesis rates and proton translocation efficiency

  • Correlate with Prochlorococcus' adaptation to nutrient-limited environments

These approaches can help understand how atpG contributes to the streamlined regulation that represents an adaptive mechanism in Prochlorococcus .

How do post-translational modifications affect atpG function?

While specific information about post-translational modifications (PTMs) of atpG in Prochlorococcus is limited in the search results, researchers can explore this area through:

• PTM detection methods:

  • Phosphoproteomic analysis using LC-MS/MS

  • Western blotting with phospho-specific antibodies

  • Phos-tag SDS-PAGE for mobility shift detection

• Functional significance assessment:

  • Site-directed mutagenesis of potential modification sites

  • In vitro modification using purified kinases/phosphatases

  • Correlation with ATP synthase activity under varying conditions

• Regulatory context:

  • Prochlorococcus shows unusual regulatory patterns, including the absence of phosphorylation in the PII protein under conditions where other cyanobacteria show this modification

  • Similar unusual PTM patterns might exist for atpG, contributing to the unique metabolic adaptations of Prochlorococcus

This research direction is particularly relevant given Prochlorococcus' streamlined regulatory mechanisms, which may extend to post-translational control of ATP synthase function.

What are the specific challenges in working with membrane proteins like atpG?

Researchers face several technical challenges when working with the membrane-associated atpG protein:

• Expression obstacles:

  • Potential toxicity to host cells due to membrane insertion

  • Protein aggregation and inclusion body formation

  • Solution: Co-expression with chaperones (DnaK, DnaJ, GrpE) can significantly improve yields of correctly folded protein

• Solubilization considerations:

  • Selecting appropriate detergents that maintain protein structure

  • Balancing solubilization efficiency with retention of native conformation

  • Solution: Screening multiple detergents including n-dodecyl β-D-maltoside, digitonin, and amphipols

• Functional assessment complexities:

  • Need for reconstitution into lipid bilayers for activity assays

  • Difficulties in measuring functional parameters in isolation from the complete ATP synthase complex

  • Solution: Development of minimal functional systems using only essential subunits

• Structural analysis limitations:

  • Challenges in obtaining high-resolution structural data for membrane proteins

  • Solution: Utilize complementary approaches including cryo-EM, solid-state NMR, and computational modeling

These challenges require optimization of every step from gene design through purification and characterization.

How can researchers address protein aggregation during recombinant atpG production?

Protein aggregation represents a significant challenge in recombinant atpG production. Based on successful approaches with similar membrane proteins, researchers should consider:

• Expression optimization:

  • Lower expression temperatures (16-20°C)

  • Reduced inducer concentration (0.1-0.5 mM IPTG instead of 1.0 mM)

  • Shorter induction times (30 minutes as recommended)

• Solubility enhancement:

  • Fusion tags: MBP tag has proven effective for membrane proteins similar to atpG

  • Co-expression with molecular chaperones using systems like pOFXT7KJE3

  • Addition of stabilizing agents (glycerol, specific ions) to growth media

• Extraction strategies:

  • Carefully optimized lysis buffers with protease inhibitors

  • Gentle solubilization using mild detergents

  • Avoiding harsh sonication conditions

• Purification approaches:

  • Size exclusion chromatography to separate aggregates

  • On-column refolding during affinity purification

  • Addition of stabilizing agents in purification buffers

Implementing these strategies in combination can significantly reduce aggregation issues and improve the yield of functional protein.

What methodological advances might improve structural studies of atpG?

Recent technological developments offer new possibilities for structural characterization of challenging membrane proteins like atpG:

• Cryo-electron microscopy advances:

  • Single-particle analysis with direct electron detectors

  • Phase plate technology for improved contrast

  • Focused refinement approaches for flexible regions

• Membrane mimetic systems:

  • Nanodiscs composed of scaffold proteins and native lipids

  • Styrene-maleic acid lipid particles (SMALPs) for detergent-free extraction

  • Peptidisc technology for stabilization of membrane proteins

• Integrative structural approaches:

  • Combining low-resolution cryo-EM with computational modeling

  • Cross-linking mass spectrometry to establish distance constraints

  • Solid-state NMR for specific structural elements

• Machine learning applications:

  • Improved protein structure prediction (AlphaFold2-type approaches)

  • Enhanced image processing for cryo-EM data

  • Better molecular dynamics simulations of membrane protein behavior

These methodological advances can be applied to atpG research to gain insights into its structure-function relationships within the ATP synthase complex.

How does atpG sequence variation across Prochlorococcus ecotypes correlate with ecological adaptations?

Sequence analysis reveals interesting variations between different strains:

What are the key unanswered questions regarding atpG in Prochlorococcus marinus?

Several critical questions remain unanswered and represent important future research directions:

• Stoichiometry and organization:

  • How many copies of atpG are present in the complete ATP synthase complex?

  • What is the specific arrangement of atpG relative to other subunits?

• Regulatory mechanisms:

  • Is atpG expression regulated in response to environmental factors?

  • Does its regulation follow the unusual patterns observed for other genes in Prochlorococcus?

• Evolutionary adaptations:

  • How has atpG evolved to function optimally in the unique environmental niche of Prochlorococcus?

  • Do the sequence variations across ecotypes correlate with functional differences?

• Energy optimization:

  • How does atpG contribute to the energy efficiency of Prochlorococcus in nutrient-limited environments?

  • Is there evidence for streamlined regulation of ATP synthase similar to that observed for nitrogen metabolism?

Addressing these questions will contribute to our understanding of how Prochlorococcus, an ecologically critical marine cyanobacterium, has evolved its bioenergetic systems.

What emerging technologies will advance atpG functional characterization?

Future research on atpG will benefit from several emerging technologies:

• Single-molecule techniques:

  • High-speed atomic force microscopy to visualize ATP synthase dynamics

  • Single-molecule FRET to measure conformational changes during catalysis

  • Optical tweezers to study mechanical properties of ATP synthase

• Advanced imaging:

  • Super-resolution microscopy to localize atpG within cyanobacterial cells

  • Cryo-electron tomography of intact thylakoid membranes

  • Correlative light and electron microscopy for in situ structural analysis

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Genome-scale metabolic modeling to predict effects of atpG variations

  • Network analysis of ATP synthase interactions with other cellular components

• Synthetic biology tools:

  • CRISPR-Cas9 genome editing in marine cyanobacteria

  • Optogenetic control of ATP synthase activity

  • Creation of minimal ATP synthase systems for mechanistic studies

These technologies will enable researchers to address fundamental questions about atpG function in the context of Prochlorococcus ecology and evolution.