Recombinant Ashbya gossypii RuvB-like helicase 1 (RVB1)

What is Ashbya gossypii RuvB-like helicase 1 (RVB1) and what functional roles does it play in cellular processes?

RuvB-like helicase 1 (RVB1) in Ashbya gossypii is a highly conserved AAA+ (ATPases Associated with various cellular Activities) family protein that functions in multiple essential cellular processes. Based on homology with well-characterized RuvB-like helicases in other organisms, A. gossypii RVB1 likely participates in:

• DNA replication and repair mechanisms

• Chromatin remodeling complexes

• RNA processing pathways

• Mitotic spindle assembly

• Transcriptional regulation

RVB1 typically forms hexameric ring structures that use ATP hydrolysis to drive DNA or RNA strand separation and protein complex remodeling. In A. gossypii, RVB1 has been implicated in nuclear functions similar to those observed in the related yeast Saccharomyces cerevisiae, where it participates in essential cellular processes .

How does A. gossypii RVB1 compare structurally and functionally to RuvB helicases in other organisms?

A. gossypii RVB1 shares significant sequence and structural homology with RuvB-like helicases across different organisms, but with species-specific adaptations:

A. gossypii RVB1 likely reflects its filamentous fungal lifestyle, with potential specialized functions in multinucleated hyphal growth and development not present in unicellular yeasts. Like other eukaryotic RVB1 proteins, it typically functions together with RVB2 as part of larger protein complexes rather than independently .

What expression systems have proven most effective for producing recombinant A. gossypii RVB1?

Several expression systems have been successfully employed for recombinant production of A. gossypii RVB1, each with distinct advantages:

• Homologous expression in A. gossypii: Provides native post-translational modifications and proper folding

  • Requires use of strong promoters such as AgTEF or AgGPD

  • Expression yields can reach 248-1127 U/mL with optimized promoters

  • Best for functional studies requiring authentic protein modifications

• E. coli expression systems:

  • BL21(DE3) strains with pET-based vectors for high yield

  • Fusion tags (His6, GST, MBP) improve solubility and facilitate purification

  • Lower authenticity but higher protein yield

• S. cerevisiae expression:

  • Leverages genomic similarities between A. gossypii and S. cerevisiae

  • Provides eukaryotic post-translational modifications

  • Expression using GAL1 promoter allows controlled induction

The choice depends on research goals: homologous expression in A. gossypii provides the most authentic protein but with lower yields, while heterologous systems offer higher production efficiency but potential differences in protein modifications and activity .

What is the role of A. gossypii RVB1 in multinucleated syncytial growth compared to its function in unicellular fungi?

A. gossypii's distinctive multinucleated filamentous growth pattern creates unique requirements for RVB1 function not present in unicellular fungi like S. cerevisiae:

• Nuclear synchronization: RVB1 likely participates in coordinating the hundreds of nuclei present within the A. gossypii syncytium, potentially through chromatin remodeling complexes that regulate gene expression across multiple nuclei simultaneously.

• Septin-mediated growth regulation: Studies of A. gossypii septins suggest that nuclear division and hyphal growth are coordinated processes in which RVB1 may play a role, particularly in DNA replication timing across multiple nuclei .

• Developmental regulation: Recent studies have revealed that A. gossypii contains developmentally regulated duplicated genes that control septin organization and cell polarity, suggesting RVB1 may have specialized functions during different growth phases .

This specialized role is demonstrated by the substantial differences in phenotypes between RVB1 mutants in A. gossypii and unicellular yeasts. In A. gossypii, RVB1 likely participates in complex mechanisms that regulate the positioning of multiple nuclei along hyphae and coordinate nuclear division with hyphal extension and branching .

What experimental approaches are most effective for studying the ATPase activity of recombinant A. gossypii RVB1?

The ATPase activity of recombinant A. gossypii RVB1 can be effectively studied through multiple complementary approaches:

• Colorimetric phosphate release assays:

  • Malachite green assay for quantifying inorganic phosphate release

  • Optimal reaction conditions: 30°C, pH 7.5, 5mM MgCl₂, 1-5mM ATP

  • Controls should include heat-inactivated RVB1 and no-protein controls

• Coupled enzyme assays:

  • NADH-coupled ATPase assay (pyruvate kinase/lactate dehydrogenase system)

  • Real-time monitoring of ATP hydrolysis through NADH absorbance decrease at 340nm

  • Allows kinetic parameters (Km, Vmax) determination

• Radiolabeled ATP hydrolysis:

  • Using [γ-³²P]ATP to directly quantify ATPase activity

  • Thin-layer chromatography separation of ATP and released phosphate

  • Provides highly sensitive measurement for low enzyme concentrations

Experimental design should consider:

• RVB1 often requires RVB2 for optimal activity

• DNA/RNA substrates may significantly enhance ATPase activity

• Protein co-factors can dramatically alter enzymatic parameters

Data interpretation should account for:

• Substrate concentration effects on activity

• The impact of reaction conditions (pH, temperature, salt concentration)

• Comparison with ATPase activity of RVB1 from other species using similar methods

How can CRISPR-Cpf1 systems be optimized for precise modification of the RVB1 gene in A. gossypii?

CRISPR-Cpf1 (Cas12a) systems offer significant advantages for precise RVB1 gene editing in A. gossypii compared to CRISPR-Cas9:

• PAM sequence selection optimization:

  • Cpf1 recognizes T-rich PAM sequences (5'-TTTN-3') which occur frequently in the AT-rich A. gossypii genome

  • Target site selection within RVB1 should prioritize regions with:

    • High conservation scores (for functional domains)

    • Minimal off-target matches throughout genome

    • Optimal positioning for desired edit

• Multiplex editing strategies:

  • Cpf1 systems can process multiple crRNAs from a single transcript

  • Design crRNA arrays targeting both RVB1 and marker genes (HIS3, ADE2, TRP1, LEU2, URA3) for co-selection

  • Use donor DNA arrays for simultaneous introduction of multiple modifications

• Delivery optimization:

  • Transform A. gossypii spores (germlings) rather than mature hyphae

  • Select primary heterokaryon clones using G418 resistance

  • Generate homokaryon clones through sporulation of primary transformants

Efficiency can vary significantly based on target sequence selection, with observed editing rates ranging from 28% to 92% depending on the target site. The system allows tagging RVB1 with fluorescent proteins, introducing point mutations to study specific functional domains, or creating conditional alleles for detailed phenotypic analysis .

What are the optimal conditions for expressing and purifying functional recombinant A. gossypii RVB1?

The expression and purification of functional recombinant A. gossypii RVB1 requires careful optimization of multiple parameters:

Expression Optimization:

• Vector selection:

  • For A. gossypii expression: Integrative cassettes with strong promoters (PGPD1, PTEF, PTSA1) outperform episomal vectors

  • For E. coli expression: pET vectors with T7 promoters and fusion tags

• Culture conditions:

  • A. gossypii: 28-30°C in rich media (MA2) with 2% glucose, supplemented with 0.5% glycerol enhances protein production by ~1.5-fold

  • E. coli: Induction at OD600 0.6-0.8, 18°C overnight with 0.1-0.5mM IPTG reduces inclusion body formation

Purification Protocol:

• Cell lysis:

  • A. gossypii: Enzymatic digestion of cell wall (zymolyase) followed by gentle mechanical disruption

  • Buffer composition: 50mM Tris-HCl pH 7.5, 150mM NaCl, 10% glycerol, 1mM DTT, protease inhibitors

• Purification steps:

  • Affinity chromatography (Ni-NTA for His-tagged proteins)

  • Ion exchange chromatography (MonoQ column at pH 8.0)

  • Size exclusion chromatography (Superdex 200) to isolate hexameric complexes

• Critical factors:

  • Keep samples at 4°C throughout purification

  • Include 5mM ATP and 5mM MgCl₂ in buffers to stabilize hexameric assembly

  • Verify functional activity immediately after purification

  • Add 10% glycerol to storage buffer and store at -80°C in small aliquots

Typical yields from optimized A. gossypii expression systems range from 0.5-5 mg/L of culture, with preservation of ATPase activity requiring the presence of its binding partner RVB2 .

What assays can be employed to characterize the helicase activity of A. gossypii RVB1 in vitro?

Multiple complementary assays can effectively characterize the helicase activity of A. gossypii RVB1:

• Fluorescence-based unwinding assays:

  • Substrate: Fluorophore-quencher labeled DNA/RNA duplexes

  • Detection: Real-time monitoring of fluorescence increase upon strand separation

  • Advantages: Quantitative, continuous measurement, high sensitivity

  • Reaction conditions: 30°C, 20mM Tris-HCl pH 7.5, 50mM NaCl, 3mM MgCl₂, 2mM ATP, 1mM DTT

• Gel-based unwinding assays:

  • Substrate: ³²P-labeled or fluorescently-labeled DNA/RNA duplexes

  • Detection: Native PAGE separation of unwound versus intact substrates

  • Advantages: Visual confirmation of unwinding, can use complex substrates

  • Key controls: Heat-denatured substrate (positive control), no-ATP reaction (negative control)

• Single-molecule approaches:

  • Techniques: Magnetic tweezers or FRET-based measurements

  • Advantages: Direct observation of unwinding kinetics at single-molecule resolution

  • Measurements: Step size, processivity, force-velocity relationships

Data analysis considerations:

• RVB1 typically functions optimally with RVB2 as a heterohexamer

• Include controls testing ATPase-deficient mutants (Walker A/B mutants)

• Test various DNA structures (3' overhangs, 5' overhangs, blunt ends)

• Compare activity with established helicase standards (rates typically range from 0.5-10 bp/sec)

A comprehensive characterization requires testing different substrate structures, as RuvB-like helicases often show structure-specific preferences in their unwinding activity. Additionally, protein co-factors may significantly enhance or alter the substrate specificity of RVB1 .

How can researchers design experiments to elucidate RVB1's role in A. gossypii growth and development?

Elucidating RVB1's role in A. gossypii growth and development requires a multifaceted experimental approach:

• Conditional knockout/knockdown systems:

  • Generate RVB1 mutant strains using CRISPR-Cpf1 technology

  • Create temperature-sensitive alleles or auxin-inducible degron tags

  • Design RVB1 protein depletion under controlled conditions to observe immediate effects

  • Examine phenotypes in different developmental stages (spore germination, hyphal extension, branching, sporulation)

• Fluorescent protein fusion analysis:

  • Create C-terminal or N-terminal GFP/mCherry fusions using integrative cassettes

  • Validate functionality of fusion proteins by complementation tests

  • Use time-lapse microscopy to track RVB1 localization during:

    • Cell cycle progression across multiple nuclei

    • Hyphal growth and branching events

    • Response to environmental stressors

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation followed by mass spectrometry

  • Use proximity-labeling techniques (BioID or APEX2) for in vivo interaction mapping

  • Verify key interactions through yeast two-hybrid or split-fluorescent protein complementation

• Transcriptomic analysis:

  • Compare gene expression profiles between wild-type and RVB1-depleted strains

  • Conduct RNA-seq at different developmental stages

  • Identify pathways dysregulated upon RVB1 perturbation

• Quantitative phenotyping:

  • Measure growth parameters (hyphal extension rate, branching frequency, nuclear division rate)

  • Evaluate stress responses (heat shock, DNA damage, protein misfolding)

  • Compare developmental timing markers with related species like Ashbya aceri

This comprehensive approach can reveal how RVB1 contributes to the unique aspects of A. gossypii biology, particularly its multinucleated syncytial growth pattern and developmental transitions .

Why might recombinant A. gossypii RVB1 show different activity levels in different experimental conditions?

Variation in recombinant A. gossypii RVB1 activity across different experimental conditions can be attributed to several factors:

• Protein complex formation requirements:

  • RVB1 typically functions as part of a heterohexameric complex with RVB2

  • Absence of binding partners can reduce activity by 50-80%

  • Different experimental conditions may affect complex assembly/stability

• Post-translational modifications:

  • Expression system impacts phosphorylation patterns crucial for activity

  • A. gossypii RVB1 expressed in E. coli lacks eukaryotic modifications

  • Phosphorylation at conserved sites can alter ATPase activity by 2-3 fold

• Buffer composition effects:

  • Ionic strength variations significantly impact hexamer formation

    • Optimal activity at 50-150mM NaCl

    • Sharp decline in activity >200mM salt

  • Divalent cation requirements: Mg²⁺ (optimal 2-5mM) vs. Mn²⁺ (1-2mM)

  • ATP concentration and ATP/ADP ratio affect conformational states

• Substrate specificity considerations:

  • DNA structure preferences (forked DNA vs. blunt ends)

  • RNA interactions may require specific secondary structures

  • Protein substrates may require co-chaperones for proper presentation

• Temperature sensitivity:

  • Activity typically peaks at 30°C for A. gossypii proteins

  • Significant reduction in activity at temperatures >37°C

  • Cold-sensitivity observed below 16°C (as seen in other A. gossypii proteins)

To minimize variability, researchers should standardize reaction conditions, ensure proper complex formation, and include appropriate controls. When comparing results across studies, these variables must be carefully considered to ensure accurate interpretation of RVB1 functional data .

How can researchers troubleshoot low expression yields of recombinant A. gossypii RVB1?

Low expression yields of recombinant A. gossypii RVB1 can be addressed through a systematic troubleshooting approach:

• Promoter optimization:

  • Replace standard promoters with stronger alternatives:

    • PGPD1 and PTEF promoters have shown 8-fold improvement in expression compared to ScPGK1

    • PTSA1 promoter may provide additional enhancement for difficult proteins

  • Consider carbon source-regulated promoters that can be induced at specific time points

• Codon optimization strategies:

  • Analyze the RVB1 coding sequence for rare codons in the expression host

  • Optimize the coding sequence while maintaining key regulatory elements

  • Focus optimization on the N-terminal region which often limits translation initiation

• Expression vector design improvements:

  • Remove problematic terminator sequences (such as ScADH1 terminator which can affect replication in A. gossypii)

  • Use integrative cassettes targeting neutral loci (ADR304W or AGL034C) rather than episomal vectors

  • Include optimized secretion signals if secreted expression is desired

• Culture condition modifications:

  • Test alternative carbon sources (glycerol can increase yields by ~1.5-fold)

  • Optimize temperature (lowering to 24-26°C may improve folding)

  • Supplement with specific additives:

    • Add 0.1% Triton X-100 to reduce cell clumping

    • Include 5% glycerol to stabilize protein

    • Test metal ion supplementation (Zn²⁺, Mg²⁺)

• Fusion tag strategies:

  • Add solubility-enhancing tags (SUMO, MBP, TRX)

  • Include protease cleavage sites for tag removal

  • Test various tag positions (N-terminal vs. C-terminal)

• Host strain engineering:

  • Use strains with reduced protease activity

  • Consider co-expression of chaperones to aid folding

  • Engineer strains to overexpress transcription factors that upregulate the secretory pathway

Implementation of these strategies has been shown to increase recombinant protein yields in A. gossypii from barely detectable levels to 248-1127 U/mL in optimized systems .

What are the potential pitfalls in interpreting RVB1 function from in vitro helicase assays compared to its in vivo activity?

Interpreting RVB1 function based on in vitro helicase assays requires caution due to several potential disconnects with in vivo activity:

• Complex formation discrepancies:

  • In vivo: RVB1 functions within large multi-protein complexes (INO80, SWR1, R2TP)

  • In vitro: Assays often use purified RVB1 or RVB1-RVB2 alone

  • Impact: Activity measured in vitro may be 10-100 fold lower than physiological activity

• Substrate presentation differences:

  • In vivo: DNA is packaged into chromatin, often with specific epigenetic modifications

  • In vitro: Naked DNA or RNA substrates with simplified structures are typically used

  • Impact: Substrate preference and unwinding rates may differ dramatically

• Cellular compartmentalization effects:

  • In vivo: RVB1 functions in specific nuclear locations with controlled access to substrates

  • In vitro: Homogeneous reaction conditions lack spatial organization

  • Impact: Regulatory mechanisms dependent on localization are missing

• Post-translational modification status:

  • In vivo: Dynamic phosphorylation, acetylation, and other modifications

  • In vitro: Usually uniform modification state depending on expression system

  • Impact: Critical regulatory switches may be absent

• Concentration and stoichiometry considerations:

  • In vivo: Precisely controlled protein levels and partner ratios

  • In vitro: Often non-physiological concentrations and partner availability

  • Impact: Activity may appear different at non-physiological protein concentrations