Recombinant Candida glabrata J protein JJJ2 (JJJ2)

What is the molecular structure of Recombinant Candida glabrata J protein JJJ2?

Recombinant Candida glabrata J protein JJJ2 is a full-length protein (455 amino acids) characterized by the presence of a J domain, which is the defining feature of all J-protein family members. The protein sequence begins with MSINDVTIDT and continues through several structural motifs, ending with CQQGVIEYFS RTALG. Like other J proteins, JJJ2 likely contains the four helical segments that are characteristic of J domains, with the diagnostic HPD (histidine-proline-aspartic acid) motif positioned between helices II and III. The J domain typically has a lysine-rich surface on helix II and precisely placed interhelical contact residues that stabilize the tertiary structure .

What are the functional roles of J proteins like JJJ2 in cellular processes?

J proteins like JJJ2 function as obligate cochaperones of 70-kDa heat-shock proteins (Hsp70s). The primary function of J proteins is to stimulate the ATPase activity of Hsp70 chaperones, thereby regulating their activity in multiple cellular processes. These processes typically involve protein folding, assembly, disassembly, and translocation. The J domain, particularly the conserved HPD motif, is crucial for the interaction with Hsp70 and subsequent stimulation of its ATPase activity. This interaction allows Hsp70 to engage with client proteins and assist in their proper folding or prevent aggregation during stress conditions .

How is JJJ2 classified within the J-protein family?

While the search results don't specifically classify JJJ2, J proteins are generally categorized into classes based on their domain organization. Based on the common classification system for J proteins, JJJ2 likely belongs to one of the following classes:

• Class I: Contains a J domain, a glycine/phenylalanine-rich region, and a zinc finger-like domain

• Class II: Contains a J domain and a glycine/phenylalanine-rich region

• Class III: Contains only a J domain and other unique domains

To determine JJJ2's specific classification, researchers would need to analyze its complete domain structure beyond just the J domain .

What are the optimal expression systems for producing Recombinant Candida glabrata J protein JJJ2?

Recombinant Candida glabrata J protein JJJ2 can be successfully expressed in different systems, with the two most documented being mammalian cell cultures and E. coli. Each system offers distinct advantages:

• Mammalian cell expression (Product code: CSB-MP738799CZI): This system may provide post-translational modifications more similar to those in eukaryotic organisms, potentially yielding a protein with native-like structure and function. This is particularly relevant since Candida glabrata is a eukaryotic organism .

• E. coli expression (Product code: CSB-EP738799CZI): This prokaryotic system typically offers higher yield and cost-effectiveness, making it suitable for applications where post-translational modifications are less critical .

The choice between these systems should be determined by the specific research requirements, including need for post-translational modifications, quantity required, and downstream applications.

What purification strategies are most effective for obtaining high-purity JJJ2 protein?

While specific purification strategies for JJJ2 are not detailed in the search results, the commercially available recombinant protein achieves >85% purity as determined by SDS-PAGE. Based on standard practices for recombinant protein purification, an effective strategy would likely include:

• Affinity chromatography using a tag system (the tag type is determined during the manufacturing process according to the search results)

• Size exclusion chromatography to separate JJJ2 from contaminating proteins of different molecular weights

• Ion exchange chromatography as a potential polishing step

The specific buffers and conditions would need to be optimized based on JJJ2's isoelectric point and stability characteristics .

How can researchers verify the functional activity of purified JJJ2 protein?

To verify the functional activity of purified JJJ2, researchers should consider the following methodological approaches:

• ATPase stimulation assay: Since J proteins stimulate the ATPase activity of Hsp70s, measuring the increase in ATPase activity of a cognate Hsp70 in the presence of JJJ2 would provide direct evidence of functionality.

• Protein interaction studies: Co-immunoprecipitation or pull-down assays to confirm JJJ2's ability to physically interact with its partner Hsp70(s).

• Complementation assays: Based on the research showing that J domain fragments can rescue certain phenotypes in yeast lacking specific J proteins, researchers could test whether JJJ2 or its J domain can functionally replace other J proteins in appropriate model systems .

• Structural integrity analysis: Circular dichroism (CD) spectroscopy to confirm the helical content expected in a properly folded J domain.

How does the HPD motif in JJJ2 affect its interaction with Hsp70 chaperones?

The HPD (histidine-proline-aspartic acid) motif is critical for the functionality of J proteins. Research on J proteins has demonstrated that alterations in this motif, such as the H32Q mutation mentioned in the search results, abolish the ability of the J domain to stimulate Hsp70 ATPase activity and consequently impair J protein function. In the case of JJJ1 (another J protein), the H32Q mutation prevented rescue of the Δydj1 phenotype, highlighting the essential nature of this motif .

For JJJ2 specifically, the HPD motif is expected to:

• Form direct contacts with the ATPase domain of its cognate Hsp70(s)

• Position the J domain correctly for optimal interaction

• Facilitate conformational changes in Hsp70 that stimulate ATP hydrolysis

Researchers investigating JJJ2-Hsp70 interactions should consider site-directed mutagenesis of the HPD motif as a negative control in their experiments, as this would provide a non-functional JJJ2 variant for comparison .

What are the implications of JJJ2's subcellular localization for its function?

While the search results don't provide specific information about JJJ2's subcellular localization, this aspect is crucial for understanding its biological function. J proteins are distributed throughout cellular compartments, including the cytosol, mitochondria, endoplasmic reticulum, and nucleus, with their localization often correlating with their specific functions.

To determine JJJ2's localization, researchers could employ:

• Fluorescent protein tagging and confocal microscopy

• Subcellular fractionation followed by Western blotting

• Immunofluorescence using JJJ2-specific antibodies

Understanding JJJ2's subcellular localization would provide insights into its potential roles in compartment-specific protein quality control processes. For instance, if JJJ2 localizes to the endoplasmic reticulum, it might be involved in protein translocation or folding of secretory proteins, similar to other J proteins like Sec63 mentioned in the search results .

How do post-translational modifications affect JJJ2 function and stability?

Post-translational modifications (PTMs) can significantly impact protein function, localization, and stability. For JJJ2, potential PTMs might include:

• Phosphorylation: The JJJ2 sequence contains numerous serine, threonine, and tyrosine residues that could be phosphorylated, potentially regulating its activity or interactions.

• Ubiquitination: As a protein involved in protein quality control, JJJ2 itself might be regulated by the ubiquitin-proteasome system.

• Other modifications: Acetylation, methylation, or SUMOylation could also play roles in modulating JJJ2 function.

Research methodologies to investigate PTMs of JJJ2 would include:

• Mass spectrometry-based proteomics to identify specific modification sites

• Western blotting with modification-specific antibodies

• Comparison of protein expressed in different systems (e.g., E. coli vs. mammalian cells) to identify eukaryote-specific modifications

• Site-directed mutagenesis of potential modification sites to assess functional consequences

What are the optimal storage and handling conditions for maintaining JJJ2 stability?

According to the product information, several factors affect JJJ2 stability and shelf life:

• Temperature: For long-term storage, -20°C to -80°C is recommended. The liquid form generally maintains stability for 6 months, while the lyophilized form remains stable for up to 12 months at these temperatures.

• Working aliquots: These can be stored at 4°C for up to one week, but repeated freezing and thawing is not recommended as it can lead to protein denaturation and loss of activity.

• Reconstitution: For lyophilized protein, reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Addition of 5-50% glycerol (final concentration) is recommended for samples intended for long-term storage, with 50% being the default concentration.

• Initial preparation: Brief centrifugation prior to opening is recommended to bring contents to the bottom of the vial .

These handling protocols are essential for maintaining protein integrity and ensuring reliable experimental results.

How can researchers design experiments to study JJJ2 interactions with Hsp70 chaperones?

To study JJJ2-Hsp70 interactions, researchers can implement several complementary approaches:

• In vitro biochemical assays:

  • Surface Plasmon Resonance (SPR) to measure binding kinetics and affinity

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters of interaction

  • ATPase assays to measure JJJ2's ability to stimulate Hsp70 ATPase activity

• Structural studies:

  • X-ray crystallography or Cryo-EM of JJJ2-Hsp70 complexes

  • NMR studies for dynamic interaction information

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Cellular assays:

  • Co-immunoprecipitation to confirm interactions in cellular contexts

  • Bimolecular Fluorescence Complementation (BiFC) for visualization of interactions in living cells

  • Proximity ligation assays to detect endogenous protein interactions

• Genetic approaches:

  • Yeast two-hybrid screening to identify potential Hsp70 partners

  • Complementation assays in appropriate model systems (e.g., yeast deletion strains)

Researchers should include appropriate controls, such as JJJ2 variants with mutations in the HPD motif, which would be expected to abolish or significantly reduce Hsp70 interaction .

What methodologies are appropriate for investigating JJJ2's role in protein quality control?

To investigate JJJ2's role in protein quality control, researchers can employ several methodological approaches:

• Client protein identification:

  • Co-immunoprecipitation followed by mass spectrometry to identify proteins that associate with JJJ2

  • Proximity-based biotinylation (BioID or TurboID) to identify proximal proteins in living cells

• Functional assays:

  • Protein aggregation assays using model substrates in the presence/absence of JJJ2

  • Thermal stability assays to assess JJJ2's impact on client protein stability

  • Protein folding kinetics measurements using fluorescence-based approaches

• Genetic approaches:

  • CRISPR-Cas9 knockouts or knockdowns to assess cellular phenotypes

  • Overexpression studies to identify gain-of-function effects

  • Rescue experiments using wild-type and mutant JJJ2 variants

• Stress response studies:

  • Analysis of JJJ2 expression and activity under various stress conditions

  • Assessment of cellular resistance to proteotoxic stress in the presence/absence of JJJ2

How should researchers interpret differences between mammalian-expressed and E. coli-expressed JJJ2?

When working with both mammalian-expressed (CSB-MP738799CZI) and E. coli-expressed (CSB-EP738799CZI) JJJ2, researchers should systematically compare and interpret differences in several parameters:

• Structural differences:

  • Secondary structure content (measurable by circular dichroism)

  • Thermal stability profiles

  • Aggregation propensity

  • Hydrodynamic properties (size-exclusion chromatography profiles)

• Functional differences:

  • ATPase stimulation capacity

  • Binding affinity for Hsp70 partners

  • Interaction with client proteins

  • Activity in protein folding/unfolding assays

• Post-translational modifications:

  • Mass spectrometry analysis to identify modifications present in mammalian-expressed but absent in E. coli-expressed protein

  • Impact of these modifications on function and stability

What controls are essential when evaluating JJJ2 function in experimental settings?

When designing experiments to evaluate JJJ2 function, several controls are essential:

• Negative controls:

  • JJJ2 with HPD motif mutations (e.g., H→Q, P→A, or D→A) to abolish J domain function

  • Heat-denatured JJJ2 to control for non-specific effects

  • Buffer-only conditions to establish baseline measurements

• Positive controls:

  • Well-characterized J proteins with known functions (e.g., DnaJ/Hdj1) to validate assay performance

  • Complementation with wild-type JJJ2 in knockout/knockdown experiments

• Specificity controls:

  • J domain-only constructs to distinguish J domain-dependent from independent functions

  • Other J proteins to assess functional overlap and specificity

• Validation controls:

  • Multiple experimental approaches to confirm key findings

  • Dose-response relationships to establish concentration dependence

  • Time-course experiments to capture kinetic parameters

These controls help ensure that experimental observations are specifically attributable to JJJ2 function rather than artifacts or non-specific effects .

How can contradictory results in JJJ2 research be reconciled and interpreted?

Contradictory results are common in protein research and may arise from various factors. When facing contradictory data regarding JJJ2, researchers should systematically:

• Compare experimental conditions:

  • Protein source and expression system differences

  • Buffer composition, pH, and ionic strength variations

  • Temperature and other environmental factors

  • Presence of additives or stabilizers

• Examine methodological differences:

  • Different assay formats or detection methods

  • Variations in protein concentration or stoichiometry

  • Time-dependent effects or kinetic differences

  • Different model systems or cellular contexts

• Consider protein quality factors:

  • Batch-to-batch variation

  • Protein stability and aggregation state

  • Post-translational modifications

  • Presence of contaminants or co-purifying factors

• Analyze interaction partners:

  • Different Hsp70 partners may yield different results

  • Presence or absence of nucleotide exchange factors

  • Competition from endogenous J proteins

  • Client protein specificity

• Statistical approaches:

  • Meta-analysis of multiple independent studies

  • Power analysis to ensure adequate sample sizes

  • Appropriate statistical tests for the data type

By systematically addressing these factors, researchers can often reconcile apparently contradictory results and develop a more nuanced understanding of JJJ2 function.

What genomic and proteomic approaches could advance understanding of JJJ2 function?

Advanced genomic and proteomic approaches offer powerful tools for elucidating JJJ2 function:

• Genomic approaches:

  • CRISPR-Cas9 screening to identify genetic interactions with JJJ2

  • RNA-seq analysis to identify transcriptional changes upon JJJ2 manipulation

  • ChIP-seq to investigate potential roles in transcriptional regulation

  • Comparative genomics to examine JJJ2 conservation across fungal species

• Proteomic approaches:

  • Proximity labeling (BioID, TurboID) to identify the JJJ2 interactome

  • Quantitative proteomics to detect proteome-wide changes upon JJJ2 manipulation

  • Thermal proteome profiling to identify client proteins

  • Cross-linking mass spectrometry to map interaction surfaces

• Integrative approaches:

  • Correlation of transcriptomic and proteomic changes

  • Network analysis to position JJJ2 within cellular protein quality control systems

  • Systems biology modeling of JJJ2's role in proteostasis

These approaches would provide comprehensive insights into JJJ2's functional network and biological significance .

How might JJJ2 function differ across various stress conditions and cellular states?

J proteins often play critical roles in stress responses. To investigate how JJJ2 function might vary across different conditions:

• Stress response analysis:

  • Heat shock response: Examine JJJ2 expression, localization, and activity changes

  • Oxidative stress: Assess potential protective roles against ROS-induced protein damage

  • ER stress: Investigate involvement in the unfolded protein response

  • Nutrient deprivation: Examine roles in protein triage during starvation

• Cell cycle and differentiation:

  • Expression and activity changes during cell cycle progression

  • Roles in cell-type specific proteostasis networks

  • Involvement in developmental processes (if applicable to the model system)

• Pathological conditions:

  • Changes during infection processes

  • Roles in biofilm formation or other virulence mechanisms

  • Response to antifungal treatments

• Experimental approaches:

  • Time-course analyses during stress induction and recovery

  • Pulse-chase experiments to measure protein turnover rates

  • Live-cell imaging to track dynamic changes in localization and interactions

  • Conditional knockout or knockdown systems to assess temporal requirements

These investigations would provide insights into the context-dependent functions of JJJ2 and its role in cellular adaptation to changing environments.

What structural biology techniques would be most informative for understanding JJJ2 mechanism of action?

Several structural biology techniques could provide critical insights into JJJ2's mechanism of action:

• High-resolution structural techniques:

  • X-ray crystallography of full-length JJJ2 and complexes with Hsp70

  • Cryo-electron microscopy for visualization of larger complexes

  • NMR spectroscopy for dynamic regions and interaction mapping

  • Small-angle X-ray scattering (SAXS) for solution-state conformational information

• Dynamic and functional structural approaches:

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • Site-directed spin labeling and EPR spectroscopy for distance measurements

  • Single-molecule FRET to monitor conformational dynamics

  • Molecular dynamics simulations based on experimental structures

• Integrative structural biology:

  • Combining multiple experimental approaches with computational modeling

  • Correlating structural features with functional outcomes

  • Time-resolved structural studies to capture transient intermediates

These approaches would illuminate how JJJ2's structure enables its function as a cochaperone, particularly focusing on the J domain's interaction with Hsp70 and how this stimulates ATPase activity and subsequent chaperone function .