Recombinant Rhizopus oryzae FK506-binding protein 2B (FKBP3)

What is Recombinant Rhizopus oryzae FK506-binding protein 2B (FKBP3)?

Recombinant Rhizopus oryzae FK506-binding protein 2B (FKBP3) is a protein belonging to the FK506-binding protein family with enzymatic classification EC 5.2.1.8. This protein functions as a peptidyl-prolyl cis-trans isomerase (PPIase), also known as rotamase, which catalyzes the isomerization of peptide bonds at proline residues. The gene encoding this protein is designated as FKBP3 (synonyms: fpr3, RO3G_11675) in Rhizopus oryzae (also known as Rhizopus delemar). The protein is characterized by a partial amino acid sequence that includes functional domains responsible for its isomerase activity and potential regulatory functions . Unlike many other recombinant proteins on the market, FKBP3 exhibits specific binding capabilities and enzymatic activities that make it valuable for various research applications in molecular biology and biochemistry.

How does Rhizopus oryzae FKBP3 differ structurally and functionally from other FKBP family members?

Rhizopus oryzae FKBP3 (FK506-binding protein 2B) differs from other FKBP family members in several key aspects. While sharing the conserved PPIase domain common to all FKBPs, R. oryzae FKBP3 has a distinctive amino acid sequence that includes a region spanning from residue 20 to 209 of the full-length protein . The protein contains specific sequences such as "LKEPPTQLQVGVKKRIPASECTRKSHSGDELSMHYTGTLFDTGEKFDSSLDRNEPFVFTLGAGQVIQGWDQGLLGMCVGEKRRLVIPPHLGYGERGAGGVIPGGATLVFEVELLEIKPGKYNQKAMPVQQQQESPISFTSPSFLVSTGIIVALFLIVFKMAKKQDIAEANEKAAAATAEASTEKKEEKKE" .

Unlike some FKBPs like the 29-kDa FKBP from Pyrococcus horikoshii (PhFKBP29) which demonstrates both PPIase activity and chaperone-like protein folding functionality, R. oryzae FKBP3's primary function appears centered around its isomerase activity . Different FKBPs also vary considerably in their PPIase efficiency; for instance, the PhFKBP29 shows significantly lower PPIase activity compared to other archaeal 16- to 18-kDa FKBPs as measured through chymotrypsin-coupled assays . These structural and functional differences influence how these proteins interact with substrates and contribute to various cellular processes.

What expression systems are most effective for producing recombinant Rhizopus oryzae FKBP3?

Several expression systems have demonstrated effectiveness for producing recombinant Rhizopus oryzae FKBP3, each with distinct advantages depending on research needs. The most commonly utilized systems include:

For standard biochemical and structural studies, E. coli-based expression has proven reliable, offering high yields and simplified purification through affinity tags such as His-tags . The E. coli system allows for in vivo biotinylation using AviTag-BirA technology, which catalyzes amide linkage between biotin and the specific lysine of the AviTag sequence . For applications requiring more complex post-translational modifications or when protein solubility is problematic in bacterial systems, researchers should consider insect cell (Baculovirus) or mammalian expression systems . The specific tag type can be determined during the manufacturing process and optimized based on experimental requirements for downstream applications.

What purification challenges are specific to Rhizopus oryzae FKBP3, and how can they be overcome?

Purification of recombinant Rhizopus oryzae FKBP3 presents several challenges that require methodical approaches to overcome:

Challenge 1: Protein Solubility
Recombinant FKBP3 proteins can form inclusion bodies when overexpressed in E. coli. To address this:

• Use lower induction temperatures (16-20°C)

• Co-express with molecular chaperones

• Optimize expression conditions with 5-50% glycerol in the buffer system, which has been shown to improve protein stability

Challenge 2: Maintaining Enzymatic Activity
FKBP3's PPIase activity can be compromised during purification. Researchers should:

• Use Tris-based buffers with optimal pH 8.0 for maximum stability

• Include 6% trehalose in storage buffers to protect protein structure

• Avoid repeated freeze-thaw cycles by preparing working aliquots stored at 4°C for up to one week

Challenge 3: Purity Requirements
For functional studies, FKBP3 requires >85-90% purity. This can be achieved through:

• Initial immobilized metal affinity chromatography (IMAC) for His-tagged variants

• Sequential purification using ion exchange chromatography

• Size exclusion chromatography as a final polishing step

Reconstitution Protocol:
For optimal results, briefly centrifuge the lyophilized protein prior to opening, then reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% (with 50% being standard) improves long-term stability during storage at -20°C/-80°C .

How can the PPIase activity of Rhizopus oryzae FKBP3 be accurately measured in vitro?

Accurate measurement of the peptidyl-prolyl cis-trans isomerase (PPIase) activity of Rhizopus oryzae FKBP3 requires careful experimental design and specialized techniques:

Chymotrypsin-Coupled Assay:
The gold standard for PPIase activity determination is the chymotrypsin-coupled spectrophotometric assay. This technique measures the rate of the cis→trans isomerization of a proline-containing peptide substrate. The protocol involves:

• Preparation of oligopeptide substrates containing a proline residue (typically Suc-Ala-Xaa-Pro-Phe-pNA, where Xaa can be various amino acids)

• Pre-incubation of the substrate with purified FKBP3 at controlled temperature (15-25°C for optimal activity measurement)

• Addition of chymotrypsin, which specifically cleaves after phenylalanine in the trans conformation

• Continuous monitoring of p-nitroaniline (pNA) release at 390 nm

• Calculation of kinetic parameters (kcat/Km) to quantify enzymatic efficiency

Temperature Considerations:
Since R. oryzae is a fungal organism, temperature optimization is crucial. While some archaeal FKBPs show activity at extreme temperatures, fungal FKBPs typically have optimal activity at moderate temperatures (25-37°C). Testing activity across a temperature range (15-45°C) provides valuable insights into thermostability and temperature-dependent activity profiles .

Control Experiments:

• Include FK506 inhibition assays to confirm specificity

• Use other well-characterized FKBPs (e.g., human FKBP12) as positive controls

• Run parallel assays with heat-inactivated FKBP3 as negative controls

The PPIase activity can be quantified in terms of kcat/Km values, which for fungal FKBPs typically range from 10^5 to 10^6 M^-1s^-1, though R. oryzae FKBP3 might exhibit different efficiency compared to other family members.

What methods can be used to investigate the potential chaperone-like activity of FKBP3?

Investigation of potential chaperone-like activity in Rhizopus oryzae FKBP3 requires multiple complementary approaches that assess protein folding assistance and aggregation prevention:

1. Rhodanese Refolding Assay:
This assay measures the ability of FKBP3 to enhance the refolding yield of chemically denatured rhodanese, a model substrate:

• Denature rhodanese using guanidinium hydrochloride

• Initiate refolding by dilution into buffer containing varying concentrations of FKBP3

• Monitor recovery of rhodanese enzymatic activity over time

• Calculate refolding yield compared to spontaneous refolding and established chaperones

2. Thermal Aggregation Prevention Assay:
This technique assesses FKBP3's ability to suppress protein aggregation at elevated temperatures:

• Select model substrate proteins (e.g., citrate synthase, luciferase)

• Incubate at increasing temperatures (45-100°C) with and without FKBP3

• Monitor aggregation by light scattering at 320-360 nm

• Determine the temperature range and efficiency of aggregation suppression

3. Circular Dichroism Spectroscopy:
This method evaluates structural stabilization of substrate proteins:

• Record CD spectra of substrate proteins with and without FKBP3

• Monitor changes during thermal denaturation

• Calculate melting temperatures (Tm) and structural transitions

4. Fluorescence-Based Assays:
Using intrinsic tryptophan fluorescence or ANS (1-anilino-8-naphthalene sulfonate) binding:

• Measure changes in fluorescence intensity/emission maxima during protein unfolding

• Compare substrate protein stability with and without FKBP3

Based on studies of other FKBPs, such as PhFKBP29, R. oryzae FKBP3 might exhibit chaperone-like activity that enhances protein refolding yields and suppresses thermal aggregation across a wide temperature range . These activities would be distinct from its catalytic PPIase function and could provide valuable insights into FKBP3's physiological roles.

How can Rhizopus oryzae FKBP3 be used as a tool for studying protein folding mechanisms?

Rhizopus oryzae FKBP3 offers several valuable applications as a research tool for investigating protein folding mechanisms:

Proline Isomerization Rate-Limiting Steps:
FKBP3, with its PPIase activity, can be employed to identify proline isomerization as a rate-limiting step in protein folding:

• Compare folding kinetics of target proteins with and without FKBP3

• Identify proteins where folding acceleration occurs specifically in the presence of FKBP3

• Use site-directed mutagenesis to substitute specific prolines and determine their contribution to folding kinetics

Co-expression Systems for Enhanced Protein Production:
Similar to other FKBPs that show chaperone-like activity, R. oryzae FKBP3 can potentially enhance the soluble expression of recombinant proteins:

• Create co-expression vectors containing both the target protein and FKBP3

• Compare expression yields and solubility profiles with and without FKBP3 co-expression

• Evaluate expression in E. coli strains under various conditions (temperature, media composition)

This approach has shown promise with FKBPs like PhFKBP29, which increased production yields of soluble recombinant proteins in E. coli .

Temperature-Dependent Folding Studies:
If R. oryzae FKBP3 shares the thermal stability properties observed in some archaeal FKBPs, it can be used to study protein folding across a wide temperature range:

• Examine protein folding at elevated temperatures (45-100°C) with FKBP3 present

• Identify temperature-dependent folding pathways

• Investigate thermal adaptation mechanisms in protein folding

The ability of some FKBPs to suppress thermal protein aggregation makes them excellent tools for studying protein folding under conditions where aggregation competes with productive folding .

What is known about the role of FKBP3 in cellular processes based on research with human orthologs?

Research with human FKBP3 orthologs has revealed significant roles in cellular processes, providing insights that may inform studies of Rhizopus oryzae FKBP3:

Cell Proliferation Regulation:
Human FKBP3 has been shown to promote cell proliferation, particularly in non-small cell lung cancer (NSCLC):

• Upregulation of FKBP3 expression (both mRNA and protein) correlates with poor survival in NSCLC patients

• In vitro and in vivo experiments demonstrate that FKBP3 promotes cancer cell proliferation

• This suggests potential roles for fungal FKBP3 in growth regulation and cell cycle control

Epigenetic Regulation Pathway:
A significant mechanistic pathway has been established linking FKBP3 to histone modification:

• FKBP3 knockdown decreases histone deacetylase 2 (HDAC2) expression

• This leads to increased expression of p27, a cell cycle inhibitor

• HDAC2 modulates histone H3K4 acetylation by directly binding to the p27 promoter

• The proliferation-promoting effect of FKBP3 depends on HDAC2 and is inhibited by p27

Transcription Factor Regulation:
FKBP3 influences gene expression through interaction with transcription factors:

• FKBP3 induces HDAC2 promoter activity by inhibiting ubiquitination of transcription factor Sp1

• This reveals a regulatory mechanism where FKBP3 stabilizes transcription factors

MicroRNA Regulation:
FKBP3 is regulated by microRNAs:

• miR-145-5p acts as a regulator of FKBP3

• miR-145-5p overexpression suppresses cell proliferation, an effect abrogated by FKBP3 overexpression

• This establishes a miR-145-5p/FKBP3/Sp1/HDAC2/p27 pathway controlling cell proliferation

While these findings come from studies on human FKBP3, they suggest potential conserved functional roles that might be explored in Rhizopus oryzae FKBP3, particularly in cellular growth regulation and interaction with transcriptional machinery.

How might the structural differences between fungal and mammalian FKBP3 be exploited for antifungal drug development?

The structural differences between fungal and mammalian FKBP3 present valuable opportunities for selective antifungal drug development:

Comparative Structural Analysis:
To exploit these differences effectively, researchers should begin with detailed structural characterization:

• Determine the crystal structure of Rhizopus oryzae FKBP3 using X-ray crystallography

• Compare with existing mammalian FKBP structures to identify:

  • Unique binding pockets in the fungal protein

  • Differences in active site architecture

  • Surface charge distribution variations

  • Fungal-specific structural motifs

• Perform molecular dynamics simulations to understand dynamic differences in protein flexibility and ligand binding

Structure-Based Drug Design Approach:
With structural insights, researchers can pursue targeted drug development:

• Use in silico screening of compound libraries against unique fungal FKBP3 binding sites

• Design competition assays measuring:

  • Binding affinity (Kd) to fungal versus mammalian FKBP3

  • Inhibition of PPIase activity (IC50 values) with selectivity ratios

  • Effects on potential chaperone-like activity

• Develop compounds that exploit the amino acid sequence differences in regions like "LKEPPTQLQVGVKKRIPASECTRKSHSGDELSMHYTGTLFDTGEKFDSSLDRNEPFVFTLGAGQVIQGWDQGLLGMCVGEKRRLVIPPHLGYGERGAGGVIPGGATLVFEVELLEIKPGK" which may contain fungal-specific elements

Functional Validation of Target:
Before investing extensively in drug development, validate FKBP3 as an antifungal target:

• Develop knockout or knockdown models in Rhizopus to assess viability and phenotypic changes

• Evaluate whether FKBP3 inhibition affects fungal growth, spore germination, or hyphal development

• Test whether inhibition sensitizes fungi to existing antifungals, potentially enabling combination therapy

Given the established role of mammalian FKBP3 in cell proliferation pathways , targeting the fungal ortholog could disrupt critical growth processes while achieving selectivity through structural differences.

What experimental approaches would best elucidate the specific cellular functions of FKBP3 in Rhizopus oryzae?

Elucidating the specific cellular functions of FKBP3 in Rhizopus oryzae requires a multi-faceted experimental approach combining molecular genetics, biochemistry, and advanced imaging techniques:

Gene Manipulation Strategies:

• CRISPR-Cas9 Gene Editing

  • Generate FKBP3 knockout strains

  • Create strains with point mutations in catalytic residues to separate enzymatic from scaffolding functions

  • Develop conditionally expressed FKBP3 variants using inducible promoters

• Protein Localization Studies

  • Generate fluorescently tagged FKBP3 constructs (GFP/mCherry fusion proteins)

  • Perform live-cell imaging to track subcellular localization

  • Use time-lapse microscopy during different growth phases and stress conditions

  • Conduct co-localization studies with markers for various cellular compartments

Protein Interaction Networks:

• Immunoprecipitation-Mass Spectrometry (IP-MS)

  • Identify FKBP3 binding partners using tagged constructs

  • Compare interactomes under different conditions (growth phases, stress)

  • Validate key interactions using techniques like bimolecular fluorescence complementation (BiFC)

• Chromatin Immunoprecipitation (ChIP)

  • Investigate whether FKBP3 associates with chromatin, similar to human FKBP3

  • Perform ChIP-seq to identify genomic binding sites

  • Analyze binding to promoters of genes involved in cell cycle regulation

Functional Characterization:

• Transcriptome Analysis

  • Compare RNA-seq profiles between wild-type and FKBP3 knockout/knockdown strains

  • Identify differentially expressed genes to reveal pathways regulated by FKBP3

  • Analyze under normal and stress conditions (temperature, nutrient limitation, cell wall stress)

• Epigenetic Studies

  • Assess histone modifications (particularly H3K4 acetylation) in FKBP3 mutants

  • Investigate potential interactions with histone deacetylases like HDAC2

  • Examine global chromatin accessibility using ATAC-seq

• Phenotypic Characterization

  • Evaluate growth rates, morphology, and development in mutant strains

  • Test stress tolerance (thermal, oxidative, osmotic)

  • Analyze cell cycle progression using flow cytometry

  • Assess protein folding capacity under stress conditions

This comprehensive approach would establish whether Rhizopus oryzae FKBP3 functions similarly to mammalian FKBP3 in regulating transcription and cell proliferation , while potentially revealing fungal-specific functions related to its peptidyl-prolyl isomerase activity or chaperone-like functions observed in other FKBPs .

What are the optimal storage and handling conditions for maintaining FKBP3 stability and activity?

Maintaining the stability and activity of recombinant Rhizopus oryzae FKBP3 requires careful attention to storage and handling conditions:

Optimal Storage Conditions:

Reconstitution Protocol:

• Briefly centrifuge the lyophilized protein vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is standard for maximum stability)

• Prepare small working aliquots to avoid repeated freeze-thaw cycles

Critical Stability Factors:

• pH Sensitivity: Maintain pH 8.0 for optimal stability; significant activity loss occurs outside pH 7.0-8.5

• Temperature Sensitivity: Avoid room temperature storage; activity decreases rapidly above 4°C for reconstituted protein

• Freeze-Thaw Cycles: Limit to absolute minimum; each cycle can reduce activity by 10-15%

• Oxidation Prevention: Include reducing agents such as DTT (0.5-1 mM) for proteins with critical cysteine residues

Activity Preservation for Functional Studies:
For experiments requiring PPIase activity measurement:

• Use freshly thawed aliquots whenever possible

• Pre-incubate for 15-30 minutes at the experimental temperature before activity assays

• Include control reactions with known activity standards to normalize for any potential activity loss

• Store enzyme separately from substrates until immediately before use

Following these guidelines ensures maximum retention of FKBP3's structural integrity and catalytic activity for research applications.

What considerations are important when designing experimental controls for studies involving FKBP3?

Designing robust experimental controls is critical for studies involving Rhizopus oryzae FKBP3 to ensure valid and reproducible results:

Enzymatic Activity Controls:

• Positive Controls:

  • Include commercially available FKBPs with well-characterized activity (e.g., human FKBP12)

  • Use previously established active batches of R. oryzae FKBP3 as internal standards

  • Run parallel assays with cyclophilin A, another PPIase family member, to distinguish family-specific effects

• Negative Controls:

  • Heat-inactivated FKBP3 (95°C for 10 minutes) to maintain protein mass but destroy activity

  • Site-directed mutants with substitutions in catalytic residues

  • Reactions with specific inhibitors like FK506 to confirm specificity of observed effects

Expression System Controls:

When studying FKBP3 expression or co-expression:

• Empty vector controls must match the backbone used for FKBP3 expression

• Non-functional FKBP3 mutant expression to control for metabolic burden

• Expression of unrelated proteins of similar size to control for generic overexpression effects

• For tagged proteins, include both N-terminal and C-terminal tag positions to account for tag interference

Cellular Function Studies:

For investigating FKBP3's roles in cellular processes:

• Gene Manipulation Controls:

  • Include both knockout/knockdown and complementation models to confirm specificity

  • Use scrambled siRNA or non-targeting guide RNA controls for RNAi or CRISPR approaches

  • Introduce wild-type gene back into knockout backgrounds to verify phenotype rescue

• Interaction Studies:

  • Include IgG controls for immunoprecipitation experiments

  • Use tag-only constructs for pull-down experiments

  • Perform reverse co-immunoprecipitation to validate interactions

  • Include known non-interacting proteins as negative controls

Cross-Contamination Prevention:

When working with multiple FKBP family members:

• Verify specificity of antibodies against different FKBP paralogs

• Include parallel experiments with closely related FKBP family members

• Use unique epitope tags to distinguish between family members in co-expression studies

These comprehensive controls account for the various confounding factors that might affect experimental outcomes when studying FKBP3, ensuring that observed effects can be confidently attributed to the protein's specific activity or function.

What are common issues encountered when working with recombinant FKBP3, and how can they be resolved?

Researchers commonly encounter several challenges when working with recombinant Rhizopus oryzae FKBP3. These issues and their solutions are outlined below:

1. Low Expression Yields:

2. Solubility Issues:

3. Loss of Enzymatic Activity:

4. Protein Aggregation During Storage:

To resolve aggregation issues:

• Add 5-50% glycerol to storage buffers

• Include 6% trehalose as a stabilizing agent

• Store at -80°C in small aliquots to prevent freeze-thaw cycles

• Filter protein solutions through 0.22 μm membranes before storage

• Consider adding non-ionic detergents at low concentrations (0.01%)

5. Inconsistent Assay Results:

For more reproducible enzymatic assays:

• Standardize protein concentration using quantitative methods (Bradford/BCA)

• Control temperature strictly during PPIase assays

• Use internal standards for normalization between experiments

• Prepare fresh substrate solutions for each experiment

• Account for auto-hydrolysis rates of chromogenic substrates in control reactions

These troubleshooting approaches address the most common challenges researchers face when working with recombinant FKBP3, improving experimental outcomes and data reliability.

How can researchers address contradictory findings regarding FKBP3 function across different experimental systems?

Addressing contradictory findings regarding FKBP3 function across different experimental systems requires a systematic approach to identify sources of variation and resolve discrepancies:

Systematic Comparative Analysis Framework:

• Source Organism Considerations:

  • Explicitly compare fungal (Rhizopus oryzae) FKBP3 with mammalian orthologs

  • Account for evolutionary divergence in protein function

  • Identify conserved versus species-specific domains that might explain functional differences

  • Consider that findings from human FKBP3 studies, such as its role in HDAC2 regulation, may not directly translate to fungal systems

• Expression System Variables:

  • Compare protein functionality when expressed in different systems:

    • Prokaryotic (E. coli) vs. eukaryotic (yeast, insect, mammalian cells)

    • Conduct side-by-side activity assays using protein from different expression systems

    • Assess post-translational modifications that might affect function

  • Standardize purification methods to minimize variability

• Methodological Harmonization:

  • Implement standardized protocols across laboratories:

    • Use consistent buffer systems and assay conditions

    • Employ the same substrate concentrations and sources

    • Standardize enzyme:substrate ratios

  • Perform inter-laboratory validation studies with identical protein batches

Reconciliation Strategies for Contradictory Data:

• Direct Comparison Experiments:

  • Design experiments specifically to test contradictory findings:

    • Use multiple methodologies to measure the same parameter

    • Include positive and negative controls relevant to each contradictory finding

    • Test dose-response relationships rather than single concentrations

• Context-Dependent Function Analysis:

  • Systematically vary experimental conditions to determine context-dependency:

    • Temperature ranges (15-45°C for enzymatic studies)

    • pH gradients (6.0-9.0)

    • Ionic strength variations

    • Presence/absence of specific cofactors or binding partners

• Structural and Functional Domain Mapping:

  • Generate domain deletion constructs to identify regions responsible for discrepant functions

  • Create chimeric proteins between different FKBP family members

  • Use site-directed mutagenesis to target specific residues implicated in contradictory functions

Integrated Data Analysis Approach:

To synthesize contradictory findings:

• Develop a unified model that accommodates context-dependent functions

• Distinguish between direct effects and downstream consequences

• Consider potential moonlighting functions of FKBP3 beyond its canonical PPIase activity

• Map contradictions to specific structural features or experimental conditions

• Acknowledge limitations of each experimental system when interpreting results

This comprehensive approach helps researchers navigate contradictory findings by identifying the specific conditions under which particular functions predominate, creating a more nuanced understanding of FKBP3's multifunctional nature across different biological contexts.

What emerging technologies could advance our understanding of FKBP3 structure-function relationships?

Several cutting-edge technologies show promise for deepening our understanding of FKBP3 structure-function relationships:

Cryo-Electron Microscopy (Cryo-EM):
Cryo-EM offers revolutionary potential for visualizing FKBP3:

• Enables visualization of FKBP3 in complex with partner proteins without crystallization

• Allows observation of multiple conformational states simultaneously

• Provides insights into dynamic processes like substrate binding and release

• Can capture transient interaction states during catalysis

AlphaFold2 and Deep Learning Approaches:
AI-driven structure prediction and analysis tools offer new perspectives:

• Generate highly accurate structural models of FKBP3 variants and complexes

• Predict effects of mutations on protein stability and function

• Model protein-protein interaction interfaces with unprecedented accuracy

• Enable virtual screening of millions of potential ligands for structure-based drug design

Single-Molecule Biophysics:
These techniques allow direct observation of individual FKBP3 molecules:

• Single-molecule FRET to measure conformational changes during catalysis

• Optical tweezers to study mechanical forces in protein folding assisted by FKBP3

• Magnetic tweezers to investigate protein-protein interactions at the single-molecule level

• These approaches can reveal heterogeneity in FKBP3 behavior masked in ensemble measurements

Native Mass Spectrometry:
Advanced MS techniques provide insights into FKBP3 complexes:

• Characterize stoichiometry and stability of FKBP3 protein complexes

• Identify post-translational modifications affecting function

• Monitor conformational dynamics in solution

• Detect weak or transient interactions that may be lost in traditional techniques

Time-Resolved Crystallography:
This emerging approach could revolutionize our understanding of FKBP3 catalysis:

• Capture structural snapshots during the PPIase reaction

• Visualize structural transitions during substrate binding and product release

• Provide direct evidence for the catalytic mechanism

• Enable structure-based design of selective inhibitors

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):
HDX-MS offers insights into protein dynamics:

• Map regions of FKBP3 that undergo conformational changes upon substrate binding

• Identify allosteric networks within the protein structure

• Compare dynamics between fungal and mammalian FKBP3 to identify functional differences

• Study effects of potential inhibitors on protein dynamics

Implementation of these technologies could resolve current contradictions in FKBP3 research and establish definitive structure-function relationships that inform both basic understanding and therapeutic applications.

What potential translational applications might emerge from advanced understanding of fungal FKBP3?

Advanced understanding of fungal FKBP3 could lead to several innovative translational applications:

Antifungal Drug Development:
Research into Rhizopus oryzae FKBP3 could enable targeted antifungal strategies:

• Design of selective FKBP3 inhibitors exploiting structural differences between fungal and human orthologs

• Development of combination therapies targeting FKBP3-dependent pathways

• Creation of peptidomimetic compounds that specifically disrupt fungal FKBP3 interactions

• These approaches could be particularly valuable against Mucormycosis (caused by Rhizopus species), which has high mortality rates and limited treatment options

Protein Production Technology:
FKBP3's potential chaperone-like functions could transform biotechnology applications:

• Development of FKBP3-based solubility tags for difficult-to-express proteins

• Creation of engineered FKBP3 variants with enhanced chaperone activity

• Design of expression systems with inducible FKBP3 co-expression for pharmaceutical protein production

• These applications could significantly improve yields of therapeutic proteins and industrial enzymes

Protein Folding Disorder Therapeutics:
Insights from FKBP3 research might inform approaches to protein misfolding diseases:

• Design of small molecules that mimic or enhance FKBP3's ability to prevent protein aggregation

• Development of targeted peptides that modulate PPIase activity in specific cellular compartments

• Creation of FKBP3-inspired synthetic chaperones for preventing protein aggregation

Synthetic Biology Applications:
Engineered FKBP3 variants could serve as components in synthetic biology circuits:

• Creation of inducible protein-protein interaction systems based on FKBP domains

• Development of protein switches controlled by small-molecule binding to FKBP3

• Design of post-translational control systems for synthetic gene networks

• Integration into biosensors for detecting specific environmental conditions

Agricultural Applications:
Understanding fungal FKBP3 could benefit agriculture:

• Development of environmentally friendly fungicides targeting pathogenic fungi

• Creation of diagnostic tools for early detection of fungal pathogens

• Engineering of crops with enhanced resistance to fungal infections through targeted inhibition of fungal FKBP3

Research Tools:
Engineered FKBP3 variants could serve as valuable research reagents:

• Development of FKBP3-based tags for protein purification under native conditions

• Creation of split-FKBP3 complementation systems for studying protein-protein interactions

• Design of FKBP3-based optogenetic tools for controlling protein function with light

These diverse applications highlight the potential impact of advancing our understanding of fungal FKBP3 beyond basic science, with implications for medicine, biotechnology, agriculture, and research methodology.