Recombinant Oryza sativa subsp. japonica Probable calcium-binding protein CML29 (CML29)

What is CML29 and how is it classified within the calcium-binding protein family?

CML29 is a calcium-binding protein belonging to the calmodulin-like (CML) family in rice (Oryza sativa). CMLs are characterized by their EF-hand motifs that facilitate calcium binding but lack additional functional domains that are present in other calcium-sensing proteins. CML29 is part of a larger multigene family of calcium sensors in rice that has been comprehensively analyzed along with other CaM and CML proteins . Unlike the canonical CaM proteins that typically have four EF-hand motifs, CML proteins can have varying numbers of these calcium-binding domains. Based on structural analyses of similar CMLs, these proteins participate in calcium signaling pathways that are essential for various cellular processes in plants .

How is CML29 structurally different from canonical calmodulin?

While both CML29 and canonical calmodulins possess EF-hand motifs for calcium binding, there are significant structural differences between them. Standard plant calmodulins are highly conserved proteins with four EF-hand motifs, whereas CML proteins like CML29 can have varying numbers of these domains. Analysis of similar CML proteins has shown that they may contain two to four EF-hand motifs, with varying calcium-binding affinities . Additionally, CMLs often exhibit more sequence divergence compared to the highly conserved CaM proteins, which typically share >90% sequence identity across species .

The three-dimensional structure of CML proteins can be predicted through homology modeling, as demonstrated for other CMLs. These analyses involve using the Phyre 2 Protein Homology/Analogy Recognition Engine to construct protein 3D structures, followed by verification of stereochemical quality using tools like the PROCHECK server . Similar methodologies can be applied to study CML29's structure.

What cellular functions does CML29 participate in?

CML29, like other calmodulin-like proteins, is involved in calcium signaling pathways that regulate various cellular processes in rice. Based on studies of related CMLs, these proteins play diverse roles in plant growth, development, and stress responses . CMLs act as calcium sensors, undergoing conformational changes upon binding calcium ions, which enables them to interact with and regulate target proteins.

Expression analysis of similar CMLs has revealed that many of these proteins are induced in response to biotic and abiotic stresses. For example, some CMLs have been shown to play roles in plant defense during herbivory . By extrapolation, CML29 may have specialized functions in calcium-mediated signaling pathways related to specific developmental processes or stress responses in rice.

What expression systems are optimal for recombinant production of rice CML29?

The optimal expression system for recombinant CML29 production is Escherichia coli, particularly strains designed for protein expression such as BL21(DE3)pLysS. This approach has been successfully employed for related calcium-binding proteins . The methodology typically involves:

• Cloning the CML29 coding sequence into a suitable expression vector (e.g., pET series vectors)

• Transforming the construct into E. coli expression strains

• Inducing protein expression with IPTG (isopropyl-β-d-thiogalactopyranoside)

• Growing cultures at reduced temperatures (15-25°C) after induction to enhance proper protein folding

For structural studies requiring isotope labeling, expression can be performed in minimal media supplemented with 15NH4Cl as the sole nitrogen source. As demonstrated for similar proteins, supplementation with 15N-Bio-Express-1000 medium after induction can enhance the yield of labeled protein .

What are the critical factors affecting the solubility and yield of recombinant CML29?

Several critical factors affect the solubility and yield of recombinant CML29:

• Induction conditions: Lower temperatures (15-20°C) and reduced IPTG concentrations (0.1-0.5 mM) often improve solubility.

• Expression time: Extended expression periods (8-10 hours or overnight) at lower temperatures can enhance proper folding.

• Media composition: Enriched media such as LB or 2xYT typically yield higher biomass, but minimal media may be necessary for isotope labeling.

• Codon optimization: Adapting codons for efficient translation in E. coli can significantly improve expression.

• Calcium availability: Presence of calcium in the growth medium may stabilize the protein structure.

Research on similar CYPs from rice has demonstrated that optimization of these factors can enable characterization of previously recalcitrant proteins and discovery of additional activities . Applying these principles to CML29 expression would likely yield similar improvements.

What purification strategy yields the highest purity for recombinant CML29?

The most effective purification strategy for recombinant CML29 typically involves:

• Initial clarification: Cell lysis followed by centrifugation to remove cellular debris.

• Affinity chromatography: If expressed with a His-tag, nickel or cobalt affinity chromatography provides high selectivity.

• Calcium-dependent chromatography: Phenyl-Sepharose hydrophobic interaction chromatography exploits the calcium-induced conformational changes, allowing purification based on the exposure of hydrophobic regions.

• Size exclusion chromatography: A final polishing step to remove aggregates and ensure homogeneity.

For CML proteins without affinity tags, calcium-dependent hydrophobic interaction chromatography has been successfully employed. This technique takes advantage of the conformational change that occurs when calcium binds to these proteins, exposing hydrophobic regions that can interact with the phenyl-Sepharose matrix .

What spectroscopic techniques are most informative for studying CML29's calcium-binding properties?

Several complementary spectroscopic techniques provide valuable insights into CML29's calcium-binding properties:

• Fluorescence Spectroscopy: Using hydrophobic probes like 8-Anilino-1-naphthalenesulfonate (ANS) allows detection of calcium-induced conformational changes. A typical protocol involves:

  • Preparing protein samples (50 μM) in appropriate buffer (e.g., 25 mM HEPES, pH 7.5, 50 mM NaCl)

  • Adding ANS (500 μM) and measuring fluorescence spectra (excitation: 380 nm, emission: 400-600 nm)

  • Comparing spectra in calcium-free (EGTA) and calcium-saturated conditions

• Circular Dichroism (CD) Spectroscopy: Provides information about secondary structure elements and their changes upon calcium binding.

• Nuclear Magnetic Resonance (NMR): 1H-15N heteronuclear single-quantum coherence (HSQC) NMR offers residue-specific information about protein-calcium interactions. This requires:

  • Expression of 15N-labeled protein in minimal media

  • Data acquisition using a high-field NMR spectrometer (600 MHz or higher)

  • Comparison of spectra in apo and calcium-bound states

• Isothermal Titration Calorimetry (ITC): Provides direct measurement of calcium binding affinities, stoichiometry, and associated thermodynamic parameters.

How can researchers accurately determine calcium-binding affinities of CML29?

Determination of calcium-binding affinities for CML29 requires methodical approaches:

• Isothermal Titration Calorimetry (ITC): The gold standard for direct measurement of binding affinities.

  • Titrate calcium solution into protein sample

  • Analyze resultant thermograms to determine binding constants (Kd values)

  • Multiple binding sites can be resolved with appropriate model fitting

• Equilibrium Dialysis: Used in conjunction with atomic absorption spectroscopy to determine bound calcium.

• Fluorescence Titration: Monitoring changes in intrinsic tryptophan fluorescence or using calcium-sensitive dyes.

• NMR Titration: Gradual addition of calcium to 15N-labeled protein while monitoring chemical shift changes in HSQC spectra.

  • Preparation of samples in 10 mM Tris-HCl, pH 6.8, 25 mM KCl

  • Incremental additions of CaCl2 to apo-protein

  • Monitoring spectral changes until saturation

Similar CMLs have shown calcium-binding affinities in the nanomolar range (30-430 nM), with multiple binding sites per molecule .

What methods can identify interaction partners of CML29?

Several complementary methods can be employed to identify CML29 interaction partners:

• Yeast Two-Hybrid (Y2H) Screening:

  • Clone CML29 into a "bait" vector (e.g., pGBKT7) fused with a DNA-binding domain

  • Transform yeast cells with this construct and a cDNA library in a "prey" vector

  • Screen transformants on selective media to identify interactions

  • Confirm interactions through reporter gene activation (e.g., MEL1 with X-galactosidase overlay assay)

  • Isolate and sequence prey plasmids to identify interactors

• Pull-down Assays:

  • Express CML29 with an affinity tag (His, GST, etc.)

  • Immobilize on appropriate matrix and incubate with plant extracts

  • Elute bound proteins and identify by mass spectrometry

• Co-immunoprecipitation:

  • Use specific antibodies against CML29 or epitope tags

  • Precipitate CML29 along with interacting partners from plant extracts

  • Identify co-precipitated proteins by Western blotting or mass spectrometry

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse CML29 and candidate interactors with complementary fragments of a fluorescent protein

  • Express in plant cells and observe fluorescence restoration when interaction occurs

How does CML29 expression change under different stress conditions?

CML29 expression patterns under various stress conditions can be assessed through:

• RNA-seq Analysis: Comparison of transcriptome data from control and stress-treated plants.

• Quantitative RT-PCR: Direct measurement of CML29 transcript levels using gene-specific primers.

  • Design primers specific to CML29 coding sequence

  • Extract RNA from tissues under different stress conditions

  • Perform RT-qPCR with appropriate reference genes

  • Analyze using the 2-ΔΔCt method for relative quantification

• Promoter Analysis: Examination of the 5' upstream region (promoter) of CML29 for stress-responsive elements.

  • Extract 1-2 kb of genomic DNA sequence upstream of the start codon

  • Analyze using tools like PlantCARE to identify regulatory elements

  • Look for elements associated with stress responses (e.g., ABRE, DRE, W-box)

Based on studies of other CMLs, expression is often induced by both biotic stresses (pathogen infection, herbivory) and abiotic stresses (drought, salt, temperature extremes). Analysis of related CMLs has revealed the presence of multiple stress-responsive elements in their promoters .

What techniques can elucidate CML29's role in calcium signaling pathways?

Several techniques can be employed to understand CML29's role in calcium signaling:

• Gene Silencing/Knockout Studies:

  • Generate CML29 knockout or knockdown lines using CRISPR-Cas9 or RNAi

  • Analyze phenotypic changes under normal and stress conditions

  • Measure calcium signaling responses using calcium-sensitive fluorescent proteins

• Overexpression Studies:

  • Generate transgenic plants overexpressing CML29

  • Examine alterations in stress tolerance and development

  • Monitor changes in downstream signaling components

• Subcellular Localization:

  • Create GFP fusion constructs with CML29

  • Express in plant cells and visualize localization using confocal microscopy

  • Verify localization using cellular fractionation and Western blotting

• Calcium-dependent Protein Interactions:

  • Perform interaction studies both in the presence and absence of calcium

  • Compare binding affinities and interaction dynamics

  • Identify calcium-dependent conformational changes that affect protein interactions

How can CML29's function be compared to other CMLs in Oryza sativa?

Comparative functional analysis of CML29 with other rice CMLs involves:

• Phylogenetic Analysis:

  • Align amino acid sequences of all rice CMLs

  • Construct phylogenetic trees to establish evolutionary relationships

  • Identify closely related CMLs for functional comparison

• Expression Pattern Comparison:

  • Analyze transcriptome data across different tissues and conditions

  • Create expression heatmaps using tools like TBtools

  • Identify co-expression patterns that might indicate functional relationships

• Protein Structure Comparison:

  • Compare predicted 3D structures of different CMLs

  • Analyze EF-hand motifs and calcium-binding sites

  • Identify unique structural features of CML29

• Functional Redundancy Testing:

  • Generate single and multiple CML mutants

  • Compare phenotypes to assess functional overlap

  • Perform complementation studies with different CMLs

Table 1: Comparison of Selected Oryza sativa CML Proteins

*Percentage identity compared to typical plant CaMs

How can isotope labeling enhance structural studies of CML29?

Isotope labeling significantly enhances structural studies of CML29 through:

• Uniform 15N Labeling:

  • Grow E. coli in M9 minimal media with 15NH4Cl as the sole nitrogen source

  • Supplement with 15N-Bio-Express-1000 medium after induction

  • Use for 2D NMR experiments (1H-15N HSQC) to monitor backbone amide resonances

• 13C/15N Double Labeling:

  • Use 13C-glucose and 15NH4Cl in minimal media

  • Enables 3D NMR experiments necessary for complete structure determination

  • Allows for detailed analysis of secondary structure elements

• Selective Amino Acid Labeling:

  • Incorporate specific labeled amino acids into the protein

  • Useful for examining regions of interest (e.g., calcium-binding sites)

  • Reduces spectral complexity for large proteins

• Deuteration:

  • Grow bacteria in D2O-based media

  • Improves spectral quality for larger proteins

  • Especially valuable when combined with TROSY-based NMR methods

These labeling strategies enable detailed structural investigations that reveal calcium-induced conformational changes and provide insights into the molecular mechanisms of CML29 function.

What are the challenges in distinguishing CML29 function from other closely related CMLs?

Distinguishing the specific functions of CML29 from closely related CMLs presents several challenges:

• Functional Redundancy:

  • Multiple CMLs may have overlapping functions

  • Single gene knockouts may show subtle or no phenotypes

  • Requires creation of multiple gene knockouts to observe clear phenotypes

• Tissue/Condition Specificity:

  • Different CMLs may be expressed in the same tissues but under different conditions

  • Requires comprehensive expression profiling across developmental stages and stress conditions

  • Necessitates careful experimental design to capture specific activation patterns

• Temporal Dynamics:

  • CML29 may function at specific time points in calcium signaling

  • Requires time-resolved measurements of calcium binding and protein interactions

  • Time-course experiments with appropriate controls are essential

• Methodological Approaches:

  • Use of specific promoters for tissue/cell-type specific expression studies

  • Development of antibodies that can distinguish between closely related CMLs

  • Creation of fluorescently tagged versions for in vivo localization and dynamics

How can researchers address inconsistent calcium-binding results in experimental studies?

Inconsistent calcium-binding results can be addressed through:

• Standardization of Experimental Conditions:

  • Maintain consistent buffer composition (pH, ionic strength)

  • Use high-purity calcium solutions with verified concentrations

  • Control temperature precisely during measurements

  • Ensure protein samples are properly folded and homogeneous

• Multiple Complementary Techniques:

  • Employ different methodologies (ITC, fluorescence, NMR) to cross-validate results

  • Compare results from different approaches to identify methodological artifacts

  • Consider the limitations of each technique in data interpretation

• Careful Sample Preparation:

  • Remove all calcium from buffers using chelating agents (EGTA, EDTA)

  • Verify calcium-free state using calcium indicators

  • Ensure protein is fully folded using CD spectroscopy

  • Check for aggregation using dynamic light scattering

• Data Analysis Considerations:

  • Use appropriate binding models (cooperative vs. independent sites)

  • Account for buffer mismatch effects in ITC experiments

  • Consider potential allosteric effects between binding sites

  • Use global fitting approaches when analyzing data from multiple techniques

How can protein aggregation be prevented during recombinant CML29 expression?

Preventing protein aggregation during recombinant CML29 expression can be achieved through:

• Optimization of Expression Conditions:

  • Lower induction temperature (15-18°C)

  • Reduce IPTG concentration (0.1-0.3 mM)

  • Shorter induction time with monitoring of soluble vs. insoluble fractions

  • Addition of osmolytes (glycerol, sorbitol) to the culture medium

• Co-expression with Chaperones:

  • Co-transform with plasmids encoding molecular chaperones (GroEL/GroES, DnaK/DnaJ)

  • Induce chaperone expression before target protein induction

  • Select appropriate chaperone systems for calcium-binding proteins

• Fusion Tag Selection:

  • Use solubility-enhancing tags (MBP, SUMO, thioredoxin)

  • Position tags at N-terminus to facilitate proper folding during translation

  • Include protease cleavage sites for tag removal after purification

• Buffer Optimization:

  • Include calcium or EGTA as needed for protein stability

  • Optimize salt concentration to prevent non-specific interactions

  • Add low concentrations of non-ionic detergents if necessary

What strategies can overcome low expression yields of CML29?

Several strategies can be employed to overcome low expression yields:

• Codon Optimization:

  • Adapt codons to match E. coli codon usage preferences

  • Analyze and eliminate rare codons, especially those occurring in clusters

  • Remove potential mRNA secondary structures that may impede translation

• Expression Vector Selection:

  • Test multiple promoter systems (T7, tac, araBAD)

  • Evaluate different affinity tags for impact on expression

  • Consider vectors with tightly controlled expression to reduce toxicity

• Host Strain Optimization:

  • Test Rosetta strains for rare codon supplementation

  • Use BL21(DE3)pLysS to reduce basal expression

  • Consider C41/C43 strains designed for toxic or membrane proteins

• Culture Condition Modifications:

  • Auto-induction media for gradual protein expression

  • High cell-density cultivation strategies

  • Supplementation with amino acids and vitamins

  • Temperature shift protocols during growth phase

Similar optimization approaches have enabled successful expression of previously recalcitrant rice CYPs, demonstrating the value of systematic optimization for challenging proteins .

How can researchers validate that recombinant CML29 maintains native structure and function?

Validating the native structure and function of recombinant CML29 requires:

• Structural Validation:

  • CD spectroscopy to confirm secondary structure content

  • NMR fingerprinting to verify proper folding

  • Thermal stability assays to assess protein stability

  • Comparison with structural parameters of related native CMLs

• Functional Validation:

  • Calcium-binding assays to confirm proper ion coordination

  • Conformational change assessment using ANS fluorescence

  • Hydrophobic exposure upon calcium binding as seen in native CMLs

  • Verification of expected calcium affinities (typically in nanomolar range)

• Interaction Validation:

  • Testing interactions with known partners of related CMLs

  • Verifying calcium dependency of these interactions

  • Competitive binding assays with native protein if available

• Activity Assessment:

  • In vitro activity assays with target proteins

  • Comparison of activation/inhibition profiles with native protein

  • Functional complementation in mutant plants or yeast systems