Recombinant Lupinus luteus Protein PR-L5

What are the major PR-10 protein variants identified in Lupinus luteus?

Lupinus luteus (yellow lupine) expresses several pathogenesis-related proteins of the PR-10 class. The most well-characterized variants include LlPR-10.1A, LlPR-10.1B, and LlPR-10.2B. These proteins have been identified in yellow lupin expression libraries from uninfected roots, suggesting constitutive expression even under non-pathogenic conditions . While the PR-10 family is widely distributed across plant species, the specific variants in Lupinus luteus have distinct properties that warrant specialized research approaches. Crystal structures have been determined for some of these proteins, particularly LlPR-10.2B in complex with various ligands, contributing significantly to our understanding of their structural and functional characteristics .

What expression systems are most effective for producing recombinant Lupinus luteus PR-10 proteins?

Recombinant expression of Lupinus luteus PR-10 proteins has been successfully accomplished using Escherichia coli systems. Specifically, plasmids of the pET series (pET-3a and pET-15b) carrying the T7 promoter have proven effective for high-yield expression . The BL21(DE3)pLysS strain of E. coli is commonly employed for this purpose. For LlPR-10.1A protein, two different expression approaches have been documented:

Western blot analysis using anti-LlPR-10.1AN antibodies has confirmed identical immunochemical properties between these recombinant proteins and native LlPR-10.1A protein, validating their structural integrity .

How can circular dichroism (CD) be used to assess the structural integrity of recombinant PR-10 proteins?

Circular dichroism (CD) spectroscopy serves as a critical quality control method for assessing the structural integrity and proper folding of recombinant PR-10 proteins from Lupinus luteus. This technique has been effectively employed for various PR-10 proteins, including LlPR-10.2B, Pinus monticola PR-10.3.1, Theobroma cacao PR-10, and Hypericum perforatum Hyp-1 .

For example, CD analysis of LlPR-10.2B provides confirmation that the recombinant protein adopts the expected secondary structure elements characteristic of PR-10 proteins. The CD spectra typically show distinctive patterns that reflect the protein's α-helical and β-sheet content, which is crucial for validating the proper folding required for biological activity . Researchers should look for characteristic minima at wavelengths indicative of α-helical content (208 and 222 nm) and β-sheet structures (~215 nm) when evaluating CD spectra of these proteins.

Any significant deviations from expected CD profiles would suggest structural abnormalities that could impact function and binding properties, making this a valuable preliminary assessment before proceeding to more resource-intensive functional studies.

What ligand-binding properties do Lupinus luteus PR-10 proteins exhibit and how are they characterized?

Lupinus luteus PR-10 proteins demonstrate remarkable versatility in binding diverse ligands, with LlPR-10.2B being particularly well-characterized for its interactions with plant hormones and regulatory molecules. These proteins possess an internal cavity capable of accommodating multiple ligand molecules simultaneously, suggesting potential roles beyond simple pathogen defense .

LlPR-10.2B has been crystallized in complex with several significant ligands:

• Phytohormones: Crystal structures have been determined for LlPR-10.2B in complex with natural cytokinins (trans-zeatin) at 1.35 Å resolution and synthetic cytokinins (diphenylurea) at 1.95 Å resolution . The protein demonstrates specific binding patterns with zeatin, with the binding cavity adopting different conformations depending on the ligand .

• Melatonin complex: LlPR-10.2B can bind two molecules of melatonin within its internal cavity, plus an additional unidentified ligand near the cavity entrance that appears to be a product of melatonin transformation .

• Quaternary complex: Remarkably, LlPR-10.2B can form a 1:1:1:1 quaternary complex with melatonin and trans-zeatin, where one melatonin-binding site is substituted with trans-zeatin while the other binding site retains melatonin .

Research techniques for characterizing these binding interactions include:

Isothermal Titration Calorimetry (ITC): This technique provides quantitative measurements of binding thermodynamics between LlPR-10.2B and its ligands. ITC experiments have been conducted for LlPR-10.2B, TcPR-10, and HpHyp-1 proteins to determine binding affinities, stoichiometry, and thermodynamic parameters .

Thermal Stability Shift Assay: This approach assesses changes in protein thermal stability upon ligand binding, providing indirect evidence of interaction strength and specificity .

X-ray Crystallography: The definitive method for determining binding modes, revealing the precise atomic interactions between PR-10 proteins and their ligands. Crystal structures of LlPR-10.2B with various ligands have been determined at high resolution, allowing detailed analysis of binding pocket architecture and ligand positioning .

What structural adaptations occur in LlPR-10.2B upon binding different ligands?

The crystal structures of LlPR-10.2B in complex with different ligands reveal fascinating structural adaptations that provide insights into its functional versatility. When comparing LlPR-10.2B complexed with different cytokinins (zeatin and diphenylurea), several key structural adjustments have been observed:

• Ligand-Specific Cavity Rearrangements: The internal binding cavity of LlPR-10.2B demonstrates remarkable plasticity, undergoing conformational changes to accommodate different ligands optimally. These adjustments involve subtle shifts in amino acid side chains lining the cavity, altering both the shape and electrostatic properties of the binding site .

• Glycine-Rich Loop Flexibility: The glycine-rich loop region shows conformational variability between different ligand complexes. In the zeatin complex (resolved at 1.35 Å), this loop adopts a specific conformation that facilitates optimal interactions with the hormone's side chain, while different arrangements are observed with other ligands .

• Asymmetric Unit Contents: Crystal structures reveal different arrangements within the asymmetric unit depending on the bound ligand, suggesting that crystal packing forces may influence the final observed conformation .

• N- and C-Termini Adaptations: The terminal regions of the protein show adaptability between different ligand complexes, potentially playing roles in controlling access to the binding cavity or in protein-protein interactions .

The structural comparison between LlPR-10.2B complexes and other PR-10 homologs (particularly the apo structure of LlPR-10.2A and the PR-10-like CSBP protein in complex with zeatin) further illuminates the structural basis for ligand recognition diversity across this protein family .

These structural adaptations suggest that PR-10 proteins may function as versatile molecular reservoirs or transporters for plant hormones, adjusting their conformation to accommodate different signaling molecules as needed during plant development and stress responses.

What evidence supports the role of Lupinus luteus PR-10 proteins in phytohormone regulation?

Multiple lines of evidence suggest Lupinus luteus PR-10 proteins may function as mediators in phytohormone regulation networks:

• Cytokinin Binding: Crystal structures of LlPR-10.2B in complex with trans-zeatin at 1.35 Å resolution reveal specific binding interactions between the protein and this important cytokinin phytohormone. The detailed binding mode has been thoroughly characterized, showing how the protein's cavity can accommodate this plant growth regulator .

• Melatonin Interactions: LlPR-10.2B demonstrates the ability to bind melatonin, an emerging plant regulator with antioxidant properties. Crystallographic studies show that two melatonin molecules can bind simultaneously within the protein's internal cavity .

• Cross-Talk Between Signaling Pathways: Perhaps most significantly, LlPR-10.2B has been crystallized in a quaternary 1:1:1:1 complex containing both melatonin and trans-zeatin, suggesting a potential role in integrating different hormonal signaling pathways . This unprecedented complex implies that PR-10 proteins might serve as mediators of melatonin-cytokinin cross-talk in plants.

• Hormone Reservoir Function: The binding characteristics support the hypothesis that PR-10 proteins may act as reservoirs for cytokinins and other regulatory molecules, potentially controlling their local concentration or availability to receptors .

• Low-Affinity Melatonin Binding: Under conditions of elevated melatonin concentration, PR-10 proteins appear to function as low-affinity melatonin binders, suggesting a possible role in buffering melatonin levels in plant tissues .

These findings collectively point to a more complex role for PR-10 proteins beyond simple pathogen defense, potentially positioning them as important players in the sophisticated hormonal regulatory networks that control plant development and stress responses.

What methods are used to investigate the antifungal properties of recombinant Lupinus luteus PR proteins?

Despite the classification of PR-10 proteins as pathogenesis-related, their direct antimicrobial activity remains controversial. Antifungal assays have been conducted for several PR-10 proteins, including LlPR-10.2B, Theobroma cacao PR-10 (TcPR-10), and Hypericum perforatum Hyp-1 . The results from these studies indicated no detectable antifungal activity for any of these three proteins .

Standard methodologies for assessing antifungal properties typically involve:

• Growth Inhibition Assays: These evaluate the ability of purified recombinant PR proteins to inhibit fungal growth in vitro. Measurements include radial growth on solid media or optical density in liquid cultures.

• Spore Germination Inhibition: This assesses whether the proteins can prevent fungal spore germination, a critical step in pathogen establishment.

• Microscopic Analysis: Examination of fungal morphology after exposure to PR proteins can reveal subtle effects on hyphal structure or development.

The lack of direct antifungal activity observed for these proteins suggests that their role in plant defense may be more complex than direct antimicrobial action. They may instead function in signaling networks, hormone regulation, or other indirect defense mechanisms. This highlights the importance of investigating multiple functional aspects beyond direct antimicrobial properties when characterizing PR proteins .

What crystallization conditions are optimal for structural studies of recombinant Lupinus luteus PR proteins?

Successful crystallization of Lupinus luteus PR-10 proteins has been achieved using specific conditions that may serve as starting points for researchers attempting structural studies. For LlPR-10.2B complexes in particular, the following approaches have proven effective:

• Ligand Co-crystallization: For the LlPR-10.2B/zeatin complex, crystals were obtained through co-crystallization techniques, where the protein solution is mixed with the ligand prior to setting up crystallization trials .

• Protein Concentration: Optimal results for LlPR-10.2B have been achieved with protein concentrated to approximately 22 mg/ml after renaturation from inclusion bodies .

• Crystallization Methods: Hanging or sitting drop vapor diffusion methods have been successfully employed, with drops containing protein-ligand mixture equilibrated against reservoir solutions .

• Data Collection Strategies: High-resolution diffraction data (1.35 Å for zeatin complex and 1.95 Å for diphenylurea complex) have been collected using synchrotron radiation sources, specifically at EMBL Unit at DESY synchrotron in Hamburg and at BESSY synchrotron in Berlin .

• Structure Solution and Refinement: Molecular replacement techniques using related PR-10 structures as search models have been effective for solving the phase problem. Programs like AMoRe have been used for this purpose .

For researchers attempting to crystallize novel PR-10 variants or complexes with different ligands, these conditions provide valuable starting points that can be optimized based on the specific protein-ligand system under investigation.

How can thermal stability shift assays be optimized for studying ligand binding to PR-10 proteins?

Thermal stability shift assays (also known as differential scanning fluorimetry or Thermofluor) provide a powerful approach for investigating ligand binding to PR-10 proteins by detecting shifts in protein melting temperature (Tm) upon ligand interaction. This technique has been successfully applied to LlPR-10.2B and Hypericum perforatum Hyp-1 proteins .

Optimized methodological considerations include:

• Protein Preparation: Using highly purified protein samples (>95% purity as assessed by SDS-PAGE) at concentrations of 0.5-1.0 mg/ml in a buffer system that maintains protein stability (typically phosphate or Tris buffers at pH 7.0-8.0).

• Fluorescent Dye Selection: SYPRO Orange is commonly used as it binds to hydrophobic regions of proteins that become exposed during thermal denaturation. The dye should be used at a final concentration of 5-10× from the commercial stock.

• Ligand Titration: Test ligands (cytokinins, melatonin, or other potential binding partners) should be examined across a concentration series, typically ranging from 10 μM to 1 mM, to establish dose-dependent effects on thermal stability.

• Temperature Range and Ramp Rate: Temperature gradients from 25°C to 95°C with a ramp rate of 1°C per minute provide optimal resolution of thermal transitions.

• Data Analysis: The melting temperature (Tm) is determined from the inflection point of the fluorescence curve, with Tm shifts of ≥1.5°C generally considered significant indicators of ligand binding.

• Controls: Include both positive controls (known ligands like zeatin) and negative controls (buffer only) to validate assay performance and establish baseline Tm values.

The thermal stability shift assay provides a rapid screening method that complements more labor-intensive techniques like isothermal titration calorimetry and X-ray crystallography, allowing researchers to quickly identify potential binding partners for further in-depth characterization .

What are the critical parameters in isothermal titration calorimetry for accurate binding studies of PR-10 proteins?

Isothermal titration calorimetry (ITC) has been successfully employed to characterize the binding thermodynamics between Lupinus luteus PR-10 proteins and various ligands. This technique provides quantitative measurements of binding affinity, stoichiometry, and thermodynamic parameters (ΔH, ΔS, and ΔG). For optimal results with PR-10 proteins, the following parameters require careful consideration:

• Protein Purity and Concentration: High-purity protein preparations (>95%) at concentrations typically between 20-50 μM in the sample cell have been used for LlPR-10.2B, TcPR-10, and HpHyp-1 proteins .

• Ligand Concentration: The ligand solution in the syringe should be approximately 10-20 times more concentrated than the protein solution to ensure saturation by the end of the titration.

• Buffer Matching: Both protein and ligand solutions must be in identical buffer conditions to minimize heat effects from buffer mismatch. Typically, phosphate buffers at pH 7.0-7.5 with low salt concentrations (50-150 mM NaCl) are employed.

• Temperature Control: Experiments are typically conducted at 25°C, with precise temperature stability (±0.1°C) throughout the experiment.

• Titration Protocol: For PR-10 proteins, optimal results have been achieved with injection volumes of 2-3 μL and adequate spacing between injections (180-300 seconds) to ensure complete return to baseline.

• Data Analysis Models: For PR-10 proteins that can bind multiple ligand molecules, advanced binding models must be considered. The "multiple sites" model is often appropriate for proteins like LlPR-10.2B that can bind two or more ligand molecules .

• Control Experiments: Ligand-into-buffer titrations are essential to correct for heats of dilution, while buffer-into-protein controls help identify any artifacts from protein dilution.

When properly optimized, ITC provides invaluable information about the thermodynamic signature of binding interactions, complementing structural studies and helping elucidate the biological significance of PR-10 protein-ligand interactions .

What are the major challenges in expressing and purifying stable recombinant PR-10 proteins?

Researchers working with recombinant Lupinus luteus PR-10 proteins face several significant challenges:

• Inclusion Body Formation: As observed with LlPR-10.1A expressed using the pET-3a system, these proteins often accumulate in insoluble inclusion bodies, necessitating renaturation procedures that can be complex and may affect protein folding .

• Concentration-Dependent Aggregation: Some variants, like His-tagged LlPR-10.1A produced using pET-15b, demonstrate concentration-dependent precipitation, limiting their utility for certain applications. This variant precipitated at concentrations above 10 mg/ml, while the renatured variant from pET-3a could be concentrated to 22 mg/ml .

• Maintaining Structural Integrity: Ensuring that recombinant PR-10 proteins maintain their native fold is essential for functional studies. Circular dichroism has proven valuable for assessing structural integrity of various PR-10 proteins including LlPR-10.2B, Pinus monticola PR-10.3.1, Theobroma cacao PR-10, and Hypericum perforatum Hyp-1 .

• Ligand Binding Capacity: Preserving the ligand-binding capacity of the recombinant proteins requires careful attention to purification conditions, as the internal cavity that accommodates ligands like cytokinins and melatonin is essential for functional studies .

• Expression System Selection: While E. coli BL21(DE3)pLysS strain with pET-series plasmids has been successfully used, optimizing expression conditions for each specific PR-10 variant remains challenging and may require tailored approaches .

Understanding and addressing these challenges is crucial for obtaining high-quality protein preparations suitable for structural, biochemical, and functional characterization of these fascinating proteins.

How might the melatonin-cytokinin binding properties of PR-10 proteins inform their potential physiological roles?

The discovery that LlPR-10.2B can simultaneously bind both melatonin and trans-zeatin suggests intriguing potential physiological roles for PR-10 proteins at the intersection of multiple hormonal signaling pathways:

• Hormone Cross-Talk Mediators: The quaternary 1:1:1:1 complex (protein:melatonin:zeatin:unknown ligand) suggests PR-10 proteins may function as molecular nodes in hormone signaling networks, potentially facilitating cross-talk between melatonin and cytokinin pathways in plants .

• Hormone Reservoirs: PR-10 proteins may serve as temporary storage reservoirs for plant hormones, helping to maintain local hormone concentrations within optimal ranges. This function could be particularly important under the conditions of elevated melatonin concentration, where PR-10 proteins appear to act as low-affinity melatonin binders .

• Hormone Transport Facilitators: The ability to bind multiple hormone types suggests PR-10 proteins might facilitate hormone transport between cellular compartments or tissues, potentially regulating hormone availability to their receptors.

• Stress Response Modulators: Since both melatonin and PR-10 proteins have been implicated in plant stress responses, their interaction might represent a mechanism for fine-tuning plant adaptation to environmental challenges.

• Developmental Regulators: Cytokinins like zeatin play crucial roles in plant development, while melatonin is emerging as an important plant regulator. PR-10 proteins could help integrate these signals during key developmental transitions.

The structural details of how LlPR-10.2B accommodates both hormones simultaneously, with one melatonin-binding site substituted by trans-zeatin while another site retains melatonin , provides valuable insights for designing experiments to test these hypothetical functions in planta.

Future research should focus on validating these potential roles through genetic approaches (e.g., knockout/overexpression studies) and in vivo hormone trafficking experiments to establish the physiological significance of these intriguing binding properties.

What computational approaches can enhance our understanding of PR-10 protein function?

Computational methods offer powerful complementary approaches to experimental studies for investigating PR-10 protein structure and function:

• Molecular Dynamics Simulations: MD simulations can reveal dynamic aspects of PR-10 proteins not captured in static crystal structures, including conformational flexibility of the binding cavity, ligand entry/exit pathways, and water molecule networks that may facilitate ligand binding. For LlPR-10.2B, simulations could elucidate how the protein accommodates different ligands and adapts its structure accordingly .

• Binding Energy Calculations: Methods such as MM-PBSA (Molecular Mechanics Poisson-Boltzmann Surface Area) can provide quantitative estimates of binding free energies between PR-10 proteins and various ligands, complementing experimental ITC measurements and helping rationalize observed binding preferences.

• Virtual Screening: In silico screening of compound libraries against the PR-10 binding cavity can identify novel potential ligands, expanding our understanding of their binding specificity and potential functional roles.

• Homology Modeling: For PR-10 variants that resist crystallization, homology models based on known structures like LlPR-10.2B can provide valuable structural insights, especially when validated against experimental data from techniques like circular dichroism .

• Sequence-Structure-Function Analysis: Comparative analysis of PR-10 sequences across species, combined with available structural data, can identify conserved features essential for function versus variable regions that may confer specificity.

• Network Analysis: Systems biology approaches can integrate PR-10 proteins into broader signaling networks, particularly in the context of hormone signaling and stress responses, helping to elucidate their position in complex regulatory systems.