Recombinant Cephalosporium acremonium Tubulin beta chain (TUB2)

What is the structural composition of Cephalosporium acremonium TUB2?

Cephalosporium acremonium TUB2 encodes the beta-tubulin chain, which forms heterodimers with alpha-tubulin to constitute microtubules. Similar to other fungal beta-tubulins, it likely contains a highly conserved core domain responsible for GTP binding and hydrolysis. The protein consists of approximately 445 amino acids with a molecular weight of approximately 50-55 kDa, comparable to other fungal beta-tubulins. The C-terminal region typically contains amino acid sequences subject to posttranslational modifications that regulate microtubule dynamics and stability . Recombinant expression systems allow for the production of this protein for detailed structural and functional analyses.

How does C. acremonium TUB2 compare to beta-tubulin from other fungal species?

Fungal beta-tubulins share significant sequence homology, particularly in the GTP-binding domains and regions involved in heterodimer formation. When comparing C. acremonium TUB2 with better-characterized fungal beta-tubulins such as those from Saccharomyces cerevisiae, researchers can expect approximately 70-85% sequence identity in conserved regions. The yeast S. cerevisiae has only one beta-tubulin gene (TUB2), which facilitates mutation studies and functional analysis . Like other fungal species, C. acremonium TUB2 likely contains species-specific variations in the C-terminal region that may influence interactions with microtubule-associated proteins and posttranslational modifications. These differences can affect microtubule dynamics and stability under various environmental conditions relevant to the ecological niche of C. acremonium.

What expression systems are suitable for producing recombinant C. acremonium TUB2?

• E. coli expression: Most suitable for basic structural studies, using vectors with T7 promoters and His-tag or GST-tag for purification

• Yeast expression: S. cerevisiae or Pichia pastoris systems when proper folding and posttranslational modifications are required

• Filamentous fungal hosts: Homologous expression in Acremonium species or related filamentous fungi for native-like modifications
  For functional studies, co-expression with alpha-tubulin may be necessary to form proper heterodimers. When expressing in E. coli, researchers should optimize codons for bacterial expression and implement low-temperature induction protocols (16-18°C) to enhance protein solubility. Purification typically involves affinity chromatography followed by size exclusion chromatography to isolate properly folded protein.

What buffers and storage conditions are optimal for recombinant C. acremonium TUB2 stability?

Based on protocols for other recombinant tubulins, researchers should consider the following conditions for C. acremonium TUB2:

How can posttranslational modifications of C. acremonium TUB2 be analyzed and their functional significance determined?

Analyzing posttranslational modifications (PTMs) of C. acremonium TUB2 requires a multi-faceted approach combining mass spectrometry with functional assays. Researchers should use the following methodological framework:

• Sample preparation: Purify native tubulin from C. acremonium using affinity chromatography with anti-beta-tubulin antibodies or isolate recombinant protein from expression systems capable of introducing fungal PTMs.

• MS/MS analysis: Employ high-resolution tandem mass spectrometry following tryptic digestion to identify specific PTMs, focusing particularly on C-terminal modifications such as detyrosination and polyglutamylation .

• Site-directed mutagenesis: Create mutants of potential modification sites identified by MS/MS to assess their functional significance.

• Comparative analysis: Similar to research on human and yeast tubulins, investigate whether C. acremonium TUB2 undergoes detyrosination by carboxypeptidases similar to TMCP1/TMCP2 identified in other species .
  The functional significance of these modifications can be determined through in vitro polymerization assays comparing native and modified forms, and through complementation studies in S. cerevisiae tub2 mutants . Additionally, researchers should examine whether C. acremonium possesses specific tubulin-modifying enzymes that might generate unique modifications compared to other fungi, affecting microtubule dynamics and interactions with associated proteins.

What are the most effective methods for studying C. acremonium TUB2 mutations and their effects on microtubule formation?

To study C. acremonium TUB2 mutations and their effects on microtubule formation, researchers should consider implementing a comprehensive experimental strategy:

• Mutation design: Based on the yeast TUB2 studies, create analogous mutations in C. acremonium TUB2 through site-directed mutagenesis, targeting residues involved in GTP binding, lateral contacts, and longitudinal interactions .

• Heterologous expression systems:

  • Express mutant forms in S. cerevisiae tub2 mutants to assess complementation ability

  • Use in vitro polymerization assays with purified recombinant proteins to evaluate assembly kinetics

  • Consider temperature-sensitive mutations similar to those created in yeast (tub2-401 through tub2-405)

• Microscopy techniques:

  • Employ immunofluorescence microscopy with anti-tubulin antibodies to visualize microtubule structures

  • Use electron microscopy to analyze protofilament formation and microtubule ultrastructure

  • Implement live-cell imaging techniques if expressing fluorescently tagged mutants in fungal cells
    The mutations created in S. cerevisiae TUB2 provide an excellent template, as they demonstrated diverse effects on microtubule arrays – from complete absence of microtubules (tub2-401) to selective effects on nuclear versus cytoplasmic microtubules (tub2-402, tub2-104) . Researchers should systematically characterize how specific mutations affect GTP hydrolysis rates, polymerization dynamics, and interactions with microtubule-associated proteins in C. acremonium.

How can recombinant C. acremonium TUB2 be utilized for studying fungal-specific inhibitors and antifungal agents?

Recombinant C. acremonium TUB2 provides an excellent platform for developing fungal-specific inhibitors with potential antifungal applications through the following methodological approaches:

• High-throughput screening assays:

  • Develop in vitro polymerization assays using purified recombinant C. acremonium TUB2 to screen compound libraries

  • Implement turbidity-based or fluorescence-based assays to monitor polymerization dynamics in the presence of potential inhibitors

  • Compare inhibition profiles between fungal and human tubulins to identify fungal-specific compounds

• Structure-based drug design:

  • Use X-ray crystallography or cryo-EM to determine the structure of C. acremonium TUB2, particularly in complex with known inhibitors

  • Identify unique fungal-specific structural features or binding pockets that can be targeted

  • Apply molecular docking and in silico screening to identify compounds with high affinity for fungal-specific sites

• Validation systems:

  • Test promising compounds in fungal growth assays

  • Evaluate effects on microtubule structures in living fungal cells using microscopy techniques

  • Assess cross-resistance with existing antifungal compounds
    This research is particularly valuable as beta-tubulin remains an important target for antifungal therapies, and species-specific differences can be exploited to develop more selective antifungal agents with reduced toxicity to human cells.

What methodologies can be used to investigate interactions between C. acremonium TUB2 and microtubule-associated proteins?

Investigating interactions between C. acremonium TUB2 and microtubule-associated proteins (MAPs) requires comprehensive protein-protein interaction methodologies:

• Affinity purification techniques:

  • GST pull-down assays using recombinant TUB2 as bait to capture interacting proteins from C. acremonium extracts

  • Co-immunoprecipitation with antibodies against TUB2 followed by mass spectrometry to identify binding partners

  • Implement BioID or proximity labeling techniques by expressing TUB2 fused to a biotin ligase

• Biophysical interaction analysis:

  • Surface plasmon resonance (SPR) to measure binding kinetics with purified MAPs

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters of interactions

  • Fluorescence resonance energy transfer (FRET) for studying interactions in solution or in cells

• Functional validation:

  • In vitro microtubule assembly/disassembly assays in the presence of putative MAPs

  • Cryo-electron microscopy to visualize MAP binding to TUB2 in polymerized structures

  • Mutational analysis of TUB2 to identify residues critical for MAP interactions
    Comparing the MAP interactome of C. acremonium TUB2 with that of other fungal species will help identify conserved and species-specific interactions. This knowledge can provide insights into unique aspects of microtubule regulation in C. acremonium and potentially reveal novel targets for selective inhibition of fungal growth.

What are the most efficient protocols for purifying recombinant C. acremonium TUB2 while maintaining its polymerization activity?

To purify recombinant C. acremonium TUB2 while preserving its polymerization activity, researchers should implement a multi-step purification protocol:

• Expression optimization:

  • Use E. coli BL21(DE3) or similar strains with reduced protease activity

  • Implement low-temperature induction (16-18°C) with 0.1-0.5 mM IPTG

  • Co-express molecular chaperones (GroEL/GroES) to improve folding

• Purification steps:

  • Cell lysis in buffer containing 50 mM PIPES (pH 6.9), 1 mM EGTA, 1 mM MgCl₂, 1 mM GTP, and 1 mM β-mercaptoethanol

  • Affinity chromatography using His-tag or similar tags (ensure tag position doesn't interfere with polymerization)

  • Tag removal using specific proteases (if tag interference is observed)

  • Ion exchange chromatography to remove degraded forms

  • Size exclusion chromatography as final polishing step

• Activity preservation:

  • Maintain GTP (0.5-1 mM) throughout purification

  • Add glycerol (5-10%) to stabilize protein

  • Avoid freeze-thaw cycles by preparing single-use aliquots

  • Consider adding polymerization stabilizers like taxol at low concentrations

• Quality control:

  • Assess purity by SDS-PAGE (>95%)

  • Verify proper folding using circular dichroism

  • Confirm GTP binding capacity using isothermal titration calorimetry

  • Validate polymerization activity using turbidity assays or electron microscopy
    This protocol can yield preparations with high polymerization activity suitable for structural and functional studies of C. acremonium TUB2.

How can researchers effectively study the dynamics of C. acremonium TUB2 polymerization and depolymerization?

Studying C. acremonium TUB2 polymerization dynamics requires specialized techniques that capture the kinetic aspects of microtubule assembly and disassembly:

• In vitro kinetic assays:

  • Light scattering turbidity measurements to monitor bulk polymerization rates

  • Total internal reflection fluorescence (TIRF) microscopy with fluorescently labeled tubulin to observe single microtubule dynamics

  • Video-enhanced differential interference contrast (DIC) microscopy for label-free visualization

• Critical parameters to measure:

  • Nucleation rate

  • Elongation rate at plus and minus ends

  • Catastrophe frequency (transition from growth to rapid shrinkage)

  • Rescue frequency (transition from shrinkage to growth)

  • GTPase activity correlation with polymerization state

• Experimental conditions to vary:

  • Temperature range (10-37°C)

  • pH (6.0-7.5)

  • Divalent cation concentrations (Mg²⁺, Ca²⁺)

  • GTP/GDP ratios

  • Presence of MAPs or potential stabilizing/destabilizing compounds

• Comparative analysis:

  • Compare dynamics with other fungal tubulins to identify species-specific characteristics

  • Assess effects of posttranslational modifications on dynamic parameters

  • Evaluate how mutations in the GTP-binding pocket or lateral contact regions affect dynamics
    These methodologies will provide insights into the unique aspects of C. acremonium TUB2 polymerization dynamics, potentially revealing adaptations specific to this fungal species that could be exploited for antifungal development.

What approaches can be used to investigate the role of TUB2 in C. acremonium cell division and morphogenesis?

Investigating TUB2's role in C. acremonium cell division and morphogenesis requires integrating genetic manipulation with advanced microscopy:

• Genetic approaches:

  • Develop CRISPR-Cas9 or RNA interference systems for C. acremonium to create conditional TUB2 mutants

  • Generate fluorescent protein fusions (GFP-TUB2) for live-cell imaging

  • Create temperature-sensitive alleles similar to the yeast tub2 mutants

  • Implement promoter replacement strategies for controlled expression levels

• Microscopy techniques:

  • Time-lapse confocal microscopy to track microtubule dynamics during hyphal growth and division

  • Super-resolution microscopy (STORM, PALM) to resolve microtubule structures at high detail

  • Electron tomography to examine the ultrastructure of the spindle and cytoplasmic microtubules

  • Correlative light and electron microscopy (CLEM) to link dynamic events with ultrastructural details

• Physiological analyses:

  • Quantify growth rates, branching patterns, and nuclear distribution in TUB2 mutants

  • Examine septation and cytokinesis processes in relation to microtubule function

  • Analyze nuclear migration and positioning defects similar to those observed in yeast tub2 mutants

  • Assess effects of antimitotic drugs with known tubulin-binding properties

• Functional rescue experiments:

  • Complement C. acremonium TUB2 mutants with tubulins from related species

  • Test whether heterologous expression of C. acremonium TUB2 can rescue yeast tub2 mutations

  • Evaluate the ability of chimeric tubulins to restore normal morphogenesis
    These approaches will elucidate how TUB2 contributes to the unique aspects of cell division and morphogenesis in filamentous fungi like C. acremonium, potentially revealing fungal-specific processes that differ from those in yeast or mammals.

What are the main challenges in expressing and working with recombinant C. acremonium TUB2, and how can they be addressed?

Researchers face several technical challenges when working with recombinant C. acremonium TUB2:

• Folding and solubility issues:

  • Challenge: Beta-tubulin often forms inclusion bodies in heterologous expression systems

  • Solution: Implement fusion tags (SUMO, MBP) that enhance solubility, reduce induction temperature to 16°C, and co-express chaperones like GroEL/GroES

  • Alternative approach: Develop cell-free expression systems with controlled redox conditions

• Polymerization competence:

  • Challenge: Recombinant tubulin often lacks proper folding or post-translational modifications for normal polymerization

  • Solution: Co-express with alpha-tubulin and necessary modification enzymes; use fungal expression systems that maintain native modifications

  • Validation method: Compare polymerization kinetics with native tubulin isolated from C. acremonium

• Stability concerns:

  • Challenge: Purified tubulin rapidly loses GTP-binding capacity and polymerization activity

  • Solution: Maintain GTP in all buffers, use stabilizing agents like glycerol or sucrose, and store at -80°C in single-use aliquots

  • Innovation: Develop cryoprotectant formulations specifically optimized for fungal tubulins

• Species-specific interactions:

  • Challenge: C. acremonium TUB2 may require species-specific factors for proper folding and function

  • Solution: Identify and co-express C. acremonium-specific tubulin folding cofactors

  • Research direction: Characterize the C. acremonium tubulin folding pathway through proteomic approaches
    Addressing these challenges requires an integrated approach combining optimized expression conditions, carefully designed purification strategies, and functional validation against native tubulin where possible.

How might comparative studies between C. acremonium TUB2 and beta-tubulins from other species advance our understanding of microtubule evolution and specialization?

Comparative studies of C. acremonium TUB2 with other species' beta-tubulins can provide significant evolutionary insights:

• Phylogenetic analysis:

  • Construct comprehensive phylogenetic trees of beta-tubulins across fungal lineages

  • Identify conserved regions and species-specific variations

  • Correlate sequence differences with ecological niches and lifecycle characteristics

  • Examine selective pressures on different domains using dN/dS ratio analysis

• Structural comparisons:

  • Perform molecular modeling of C. acremonium TUB2 based on crystal structures of other tubulins

  • Identify unique structural features that may relate to fungal-specific functions

  • Compare the GTP-binding pocket and lateral contact surfaces across species

  • Analyze differences in the C-terminal region that influence interactions with regulatory proteins

• Functional assessments:

  • Compare polymerization dynamics between recombinant tubulins from different fungal species

  • Assess cross-species compatibility in heterodimer formation with alpha-tubulins

  • Test sensitivity to various microtubule-targeting compounds across species

  • Evaluate posttranslational modification patterns and their conservation

• Evolutionary implications:

  • Investigate whether C. acremonium TUB2 shows adaptations related to its ecological niche

  • Determine if certain sequence variations correlate with the evolution of filamentous growth

  • Explore the relationship between tubulin sequence and cold sensitivity observed in some fungal species

  • Examine horizontal gene transfer possibilities within fungal lineages
    These comparative approaches will contribute to our understanding of how microtubule proteins have evolved specialized functions while maintaining core structural features necessary for polymerization.

What novel research applications might emerge from studying the interactions between C. acremonium TUB2 and other cellular components?

Studying interactions between C. acremonium TUB2 and other cellular components opens several innovative research directions:

• Identification of fungal-specific microtubule regulatory networks:

  • Use proximity labeling technologies like BioID fused to TUB2 to identify novel interactors

  • Implement cross-linking mass spectrometry to capture transient interactions

  • Develop fungal-specific two-hybrid screens optimized for TUB2 interactions

  • Compare the interactome across different fungal growth phases and stress conditions

• Development of novel bioengineering applications:

  • Engineer C. acremonium TUB2 as scaffolds for enzyme immobilization

  • Develop self-assembling nanostructures based on fungal tubulin polymerization properties

  • Create biosensors utilizing TUB2 conformational changes in response to environmental signals

  • Explore the potential of fungal microtubules as templates for nanomaterial synthesis

• Insights into cellular adaptation mechanisms:

  • Investigate how TUB2 interactions change during environmental stress responses

  • Study the relationship between TUB2 and fungal cell wall synthesis machinery

  • Examine connections between microtubule dynamics and hyphal tip growth

  • Explore potential roles in fungal virulence and pathogenicity mechanisms

• Biomimetic applications:

  • Study the mechanical properties of C. acremonium microtubules for biomaterial development

  • Investigate energy-efficient aspects of fungal microtubule assembly for synthetic biology

  • Develop fungal-based self-organizing systems inspired by microtubule dynamics These research directions could lead to applications ranging from novel antifungal development strategies to biomimetic materials and nanotechnology innovations inspired by fungal microtubule properties.