Recombinant Ricinus communis Acyl-CoA-binding protein

Basic Research Questions

• What are Acyl-CoA-binding proteins (ACBPs) and what is their function in Ricinus communis?

Acyl-CoA-binding proteins (ACBPs) are a family of proteins characterized by their ability to bind acyl-CoA esters with high specificity and affinity. They contain a conserved acyl-CoA-binding (ACB) domain that facilitates this binding. In plants including Ricinus communis (castor bean), ACBPs play crucial roles in lipid metabolism, transport, and signaling pathways.

Plant ACBPs generally participate in intracellular transport of acyl-CoA esters, protection of acyl-CoA esters from hydrolysis, regulation of lipid biosynthesis and metabolism, responses to biotic and abiotic stresses, and lipid trafficking between different cellular compartments . Based on studies in other plants like Arabidopsis and rice, we can infer that Ricinus communis ACBPs likely play important roles in castor oil biosynthesis, which is rich in ricinoleic acid, a unique fatty acid that constitutes approximately 90% of castor oil.

• How are recombinant Ricinus communis ACBPs produced and purified?

Recombinant Ricinus communis ACBPs can be produced using bacterial expression systems, most commonly Escherichia coli. Commercially available recombinant Ricinus communis Acyl CoA Binding protein is expressed in E. coli with purity >90% and supplied in PBS buffer (pH 7.4) with 50% glycerol .

A general methodology for producing recombinant plant ACBPs involves:

• Cloning the ACBP gene from Ricinus communis cDNA into a suitable expression vector

• Adding a tag (commonly polyHis-tag) to facilitate purification

• Transforming the construct into E. coli expression strains (e.g., BL21(DE3))

• Inducing protein expression with IPTG

• Lysing cells and purifying the recombinant protein using affinity chromatography

• Further purification using ion-exchange chromatography or gel filtration

For instance, researchers have successfully produced and purified a recombinant polyHis-tagged N-terminal fragment of a related lipid-binding protein from Brassica napus, which could serve as a methodological reference for Ricinus communis ACBP production .

• What are the structural characteristics of Ricinus communis ACBPs?

Plant ACBPs are typically classified into four classes based on their domain architecture:

• Class I: Small ACBPs (10 kDa) containing only the ACB domain

• Class II: Ankyrin-repeat containing ACBPs with the ACB domain and ankyrin repeats

• Class III: Large ACBPs with the ACB domain and other domains

• Class IV: Kelch-motif containing ACBPs with the ACB domain and kelch motifs

The core ACB domain is highly conserved and forms a binding pocket for acyl-CoA. Recombinant N-terminal fragments of plant acyltransferases like DGAT1 can bind acyl-CoA and potentially self-associate to form dimers and tetramers . Similar properties might exist for Ricinus communis ACBPs, especially if they function in multiprotein complexes involved in lipid metabolism.

Studies of recombinant plant ACBPs have shown that the ACB domain contains approximately 90 amino acids and forms a four-α-helix bundle structure that creates a binding site for the acyl-CoA molecule . The binding pocket accommodates both the adenine nucleotide and the fatty acyl chain of the acyl-CoA molecule.

• What is the subcellular localization of Ricinus communis ACBPs?

Different ACBP classes typically show distinct subcellular localizations that correspond to their functional roles in lipid metabolism. Based on information about ACBPs from other plant species, particularly rice (Oryza sativa), we can infer potential localizations for Ricinus communis ACBPs:

In rice, the subcellular localization of OsACBPs has been determined through GFP fusion proteins:

• OsACBP1 and OsACBP2: cytosol

• OsACBP3: multi-localization

• OsACBP4 and OsACBP5: endoplasmic reticulum (ER)

• OsACBP6: peroxisomes

The localization of Ricinus communis ACBPs would need to be experimentally determined using similar methods: fusion with GFP and observation via confocal microscopy in transient expression systems. This subcellular distribution likely reflects the involvement of different ACBP isoforms in specific aspects of lipid metabolism and trafficking that occur in distinct cellular compartments.

• How do Ricinus communis ACBPs compare to ACBPs from other plant species?

Plant ACBP family composition shows similarities across species:

• Arabidopsis thaliana: Six ACBPs (AtACBP1-6)

• Oryza sativa (rice): Six ACBPs (OsACBP1-6)

• Ricinus communis likely has a similar number of ACBP genes

Phylogenetic analyses using 16 plant genomes have shown that:

• The ACBP family diversified as land plants evolved

• Classes I and IV show lineage-specific gene expansion

• Classes II and III are closely related phylogenetically

Functional comparisons indicate that:

• Rice ACBPs show ubiquitous expression with OsACBP4, OsACBP5, and OsACBP6 being stress-responsive

• Plant ACBPs generally have roles in lipid metabolism, transport, and stress responses

• Ricinus communis ACBPs might have specialized functions related to castor oil biosynthesis, particularly in handling ricinoleic acid

Binding preferences differ between plant species and within ACBP families:

• Different ACBPs have varying affinities for different acyl-CoA chain lengths

• Rice and Arabidopsis ACBPs bind acyl-CoA esters of various chain lengths with different specificities

Advanced Research Questions

• What are the specific binding affinities of Ricinus communis ACBPs for different acyl-CoA esters?

While specific binding affinities for Ricinus communis ACBPs have not been extensively characterized, insights from other plant ACBPs provide valuable reference points:

Recombinant ACBPs from Arabidopsis and rice bind acyl-CoA esters with varying affinities:

• AtACBP1 and AtACBP3 displayed high affinity to very-long-chain (VLC) species

• AtACBP3 to AtACBP6 and OsACBPs bind medium-chain species

• All AtACBPs and OsACBPs bind long-chain acyl-CoA esters at different affinities

The N-terminal fragment of BnDGAT1 (a lipid metabolism enzyme) binds acyl-CoAs in a cooperative fashion, with different affinities for different acyl-CoAs:

• Greater affinity for erucoyl-CoA (22:1cis Δ13) than oleoyl-CoA (18:1cis Δ9)

• Dissociation constants of about 17 μM for oleoyl-CoA and 2 μM for erucoyl-CoA

To determine binding affinities for Ricinus communis ACBPs, researchers could use:

• Isothermal titration calorimetry (ITC) as used for other plant ACBPs

• Lipidex-1000 binding assays

• Scatchard plot analysis to determine dissociation constants and investigate potential cooperative binding

These experiments should test binding with various acyl-CoAs relevant to castor bean metabolism, particularly ricinoleoyl-CoA, which is important in castor oil biosynthesis.

• How can I optimize expression systems for producing functional recombinant Ricinus communis ACBPs?

Several strategies can be employed to optimize recombinant ACBP production:

Expression vector selection:

• pET vectors for T7-driven expression in E. coli

• pGEX vectors for GST-fusion proteins

• Consider codon optimization for E. coli expression

Expression host optimization:

• E. coli BL21(DE3) or derivatives like Rosetta for rare codon usage

• For membrane-associated ACBPs, consider specialized E. coli strains

Induction conditions:

• Test various IPTG concentrations (0.1-1.0 mM)

• Lower temperature induction (16-25°C) to improve folding

• Extended induction times (overnight) at lower temperatures

Solubility enhancement:

• Addition of solubility tags (MBP, SUMO, TRX)

• Co-expression with chaperones

• For hydrophobic ACBPs, addition of mild detergents or lipids

Purification optimization:

• Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

• Ion exchange chromatography as a second purification step

• Size exclusion chromatography for final polishing

Functional assessment:

• Acyl-CoA binding assays (Lipidex-1000 or ITC)

• Circular dichroism to confirm proper folding

• Thermal shift assays to assess stability

A recombinant N-terminal fragment of BnDGAT1 was successfully produced as a His-tagged protein and retained acyl-CoA binding functionality, providing a methodological template .

• What role do Ricinus communis ACBPs play in lipid metabolism and castor oil biosynthesis?

Potential roles of Ricinus communis ACBPs in castor oil biosynthesis include:

Acyl-CoA transport and protection:

• Binding and transport of acyl-CoA esters between different cellular compartments

• Protection of acyl-CoA from hydrolysis, maintaining an active acyl-CoA pool

Regulation of fatty acid biosynthesis:

• Potential interaction with fatty acid synthase (FAS) and fatty acid elongation (FAE) complexes

• Regulation of feedback inhibition by maintaining appropriate acyl-CoA concentrations

Potential role in ricinoleic acid metabolism:

• Binding and transporting ricinoleoyl-CoA, the activated form of the predominant fatty acid in castor oil

• Potential interaction with oleate Δ12-hydroxylase (FAH12), which converts oleic acid to ricinoleic acid

Interaction with acyltransferases:

• Acyl-CoA binding proteins might modulate acyltransferase activity

• Potential interaction with DGAT and PDAT enzymes involved in triacylglycerol assembly

Compartmentalization of lipid metabolism:

• Different ACBPs localize to different subcellular compartments

• This might facilitate coordination of lipid metabolism between organelles

Experimental approaches to investigate these roles could include gene expression analysis in developing castor bean seeds, protein-protein interaction studies, genetic manipulation of Ricinus communis ACBPs, and lipidomic analysis of transgenic lines.

• How do environmental stresses affect the expression and function of Ricinus communis ACBPs?

Plant ACBPs generally play roles in stress responses:

• Plant ACBPs have roles in both abiotic and biotic stress responses

• They interact with lipids and proteins in these stress response pathways

In rice, certain ACBP isoforms showed stress-responsive expression:

• OsACBP4, OsACBP5, and OsACBP6 were identified as stress-responsive

• The multidomain rice ACBPs appear to be associated with stress responses

Based on this information, Ricinus communis ACBPs might be regulated by various stresses such as drought, temperature extremes, salt stress, pathogen attack, and oxidative stress.

To investigate stress effects on Ricinus communis ACBPs:

Gene expression analysis:

• qRT-PCR to measure ACBP transcript levels under various stress conditions

• RNA-seq to examine global transcriptional changes including ACBPs

Protein analysis:

• Western blotting to quantify ACBP protein levels

• Post-translational modification analysis under stress

Functional studies:

• Metabolite profiling to detect changes in lipid composition

• Overexpression/silencing studies under stress conditions

Comparative analysis:

• Compare stress responses of different ACBP isoforms

• Correlation between stress responses and subcellular localization

• What methods can be used to study the protein-protein interactions of Ricinus communis ACBPs?

Several techniques can be employed to study protein-protein interactions of Ricinus communis ACBPs:

Co-immunoprecipitation (Co-IP):

• Generate antibodies against Ricinus communis ACBPs

• Immunoprecipitate ACBPs from plant extracts and identify interacting proteins by mass spectrometry

Yeast two-hybrid (Y2H) screening:

• Use Ricinus communis ACBPs as bait proteins

• Screen against a cDNA library from castor bean, particularly from developing seeds

• Verify interactions with directed Y2H assays

Bimolecular fluorescence complementation (BiFC):

• Fuse ACBPs to one half of a split fluorescent protein

• Fuse potential interacting proteins to the complementary half

• Transient expression in plant cells and visualization via confocal microscopy

Pull-down assays:

• Express recombinant tagged ACBPs (as in commercial preparations)

• Incubate with plant extracts and isolate complexes via affinity purification

• Identify binding partners via mass spectrometry

Surface plasmon resonance (SPR):

• Immobilize purified ACBPs on sensor chips

• Flow potential interacting proteins over the surface

• Measure binding kinetics and affinities

Crosslinking studies:

• Similar to approaches showing that BnDGAT1 fragments self-associated to form a tetramer

• Use chemical crosslinkers to stabilize protein complexes

• Analyze via SDS-PAGE and mass spectrometry

These approaches would help identify proteins that interact with Ricinus communis ACBPs, potentially revealing their roles in lipid metabolism networks and stress response pathways.

• How can recombinant Ricinus communis ACBPs be used to study lipid trafficking mechanisms?

Recombinant Ricinus communis ACBPs can serve as valuable tools for studying lipid trafficking:

In vitro lipid binding assays:

• Using purified recombinant ACBPs to measure binding affinities for different lipid species

• Similar to approaches showing that recombinant ACBPs bind not only acyl-CoA but also phospholipids like PC, PA, and PE

Fluorescent lipid trafficking:

• Labeling lipids with fluorescent tags

• Using recombinant ACBPs to track lipid movement in artificial membrane systems

• Comparing trafficking efficiency with different ACBP isoforms

Reconstitution experiments:

• Incorporating recombinant ACBPs into liposomes or artificial membrane systems

• Measuring rates of lipid transfer between membrane compartments

• Determining the effects of acyl chain length and saturation on transfer rates

Competitive binding studies:

• Using recombinant ACBPs to compete with native lipid transfer systems

• Identifying specific lipid species preferentially trafficked by different ACBP isoforms

Structure-function analysis:

• Creating mutant variants of recombinant ACBPs

• Testing how specific amino acid changes affect lipid binding and transfer

• Correlating structural features with lipid trafficking capabilities

Interaction with membrane systems:

• Studying how recombinant ACBPs interact with lipid bilayers

• Determining whether ACBPs extract lipids from membranes or dock at membrane surfaces

• Investigating potential membrane fusion or remodeling activities

Plant ACBPs are involved in nonvesicular lipid transport mechanisms , and recombinant Ricinus communis ACBPs could help elucidate these pathways, particularly those related to castor oil biosynthesis.

• What are the crystallization conditions for structural studies of Ricinus communis ACBPs?

While specific crystallization conditions for Ricinus communis ACBPs are not yet reported, strategies can be developed based on successful crystallization of related plant lipid-binding proteins:

Initial screening approaches:

• Commercial sparse matrix screens (Hampton Research, Molecular Dimensions)

• Varying pH (5.0-9.0), precipitants (PEG, ammonium sulfate), and salt concentrations

• Testing at different temperatures (4°C and 20°C)

Protein preparation considerations:

• High purity (>95%) recombinant protein

• Concentration range: 5-15 mg/mL

• Buffer optimization to enhance stability (potentially PBS buffer with glycerol)

• Consider removal of His-tag if it interferes with crystallization

Optimization strategies:

• Seeding from initial microcrystals

• Additive screening

• Surface entropy reduction through mutation of surface residues

• Co-crystallization with acyl-CoA ligands to stabilize protein conformation

Data collection considerations:

• In-house X-ray source for initial screening

• Synchrotron radiation for high-resolution data collection

• Cryoprotection optimization to prevent ice formation

The successful crystallization of a recombinant polyHis-tagged N-terminal fragment of DGAT1 from Brassica napus indicates that plant lipid-binding proteins can be crystallized, suggesting Ricinus communis ACBPs might be amenable to similar approaches.

• How do post-translational modifications affect Ricinus communis ACBP function?

Potential post-translational modifications (PTMs) that might affect Ricinus communis ACBP function include:

Phosphorylation:

• Could regulate binding affinity for acyl-CoA

• Might affect protein-protein interactions

• Could regulate subcellular localization

• May be involved in stress-responsive signaling pathways

Redox modifications:

• Oxidation of cysteine residues might affect protein structure and function

• Could serve as a mechanism for sensing oxidative stress

• May regulate oligomerization (relating to self-association of lipid-binding proteins)

Ubiquitination:

• Regulation of protein turnover

• Potential non-degradative signaling roles

• Might affect protein interactions and localization

Glycosylation:

• Potentially affecting protein stability

• Might influence interaction with membranes

• Could affect protein trafficking

To study these PTMs in Ricinus communis ACBPs, researchers could:

• Use mass spectrometry to identify PTMs in native ACBPs

• Create site-directed mutants to mimic or prevent specific modifications

• Test the functional consequences on acyl-CoA binding using methods like those established for other lipid-binding proteins

• Examine conditions that induce or remove PTMs, particularly in stress responses

• Compare PTM patterns across different developmental stages, particularly during seed development when castor oil biosynthesis is active

• What are the best experimental approaches to study the physiological roles of different Ricinus communis ACBP isoforms?

Several complementary experimental approaches can be employed:

Gene expression analysis:

• Quantitative RT-PCR to analyze expression patterns of different ACBP isoforms

• RNA-seq to examine co-expression networks

• In situ hybridization to determine tissue-specific expression

• Similar to approaches used for rice ACBPs under normal growth and stress conditions

Protein localization:

• GFP fusion constructs to determine subcellular localization

• Similar to approaches used for rice ACBPs

• Immunolocalization with isoform-specific antibodies

Genetic manipulation:

• CRISPR/Cas9 gene editing to create knockout mutants

• RNAi to silence specific isoforms

• Overexpression studies

• Complementation of mutants with specific isoforms

Biochemical characterization:

• Recombinant protein production for each isoform

• In vitro binding assays to determine substrate preferences

• Similar to approaches used to study acyl-CoA binding properties of other plant proteins

Physiological analysis:

• Phenotypic characterization of mutants/transgenic plants

• Lipid profiling to detect alterations in lipid composition

• Stress response assays, relating to known roles of plant ACBPs in stress responses

Protein interaction studies:

• Yeast two-hybrid or co-immunoprecipitation to identify interacting partners

• Bimolecular fluorescence complementation (BiFC) to confirm interactions in planta

Functional complementation:

• Cross-species complementation (e.g., expressing Ricinus communis ACBPs in Arabidopsis acbp mutants)

• Similar to expressing OsACBP6 in the Arabidopsis pxa1 mutant

These approaches, particularly when used in combination, would provide comprehensive insights into the physiological roles of different Ricinus communis ACBP isoforms.

• How can I design experiments to investigate the role of Ricinus communis ACBPs in stress response pathways?

Based on the known roles of plant ACBPs in abiotic and biotic stress responses and evidence for stress-responsive expression of rice ACBPs , several experimental approaches can be designed:

Expression analysis under stress conditions:

• Expose Ricinus communis plants to various stresses (drought, salt, cold, heat, pathogens)

• Monitor expression changes of different ACBP isoforms using qRT-PCR

• Perform time-course experiments to track dynamic responses

• Compare expression in different tissues to identify tissue-specific stress responses

Transgenic approaches:

• Generate ACBP overexpression lines in Ricinus communis

• Create RNAi or CRISPR knockout lines for specific ACBPs

• Evaluate stress tolerance phenotypes

• Analyze changes in stress-responsive marker genes

Protein-level responses:

• Develop isoform-specific antibodies to track protein levels under stress

• Analyze post-translational modifications in response to stress

• Examine changes in subcellular localization during stress

Metabolite analysis:

• Lipidomic profiling under stress conditions in wild-type and ACBP-modified plants

• Analysis of stress-related metabolites (e.g., jasmonates, salicylic acid)

• Investigation of membrane lipid remodeling during stress

Protein interaction studies:

• Identify stress-specific protein interactions using techniques like:

  • Co-immunoprecipitation under stress conditions

  • Yeast two-hybrid screening with stress-induced cDNA libraries

  • Bimolecular fluorescence complementation in stressed cells

Comparative genomics:

• Compare stress responses of Ricinus communis ACBPs with those of other plants

• Identify conserved stress-responsive elements in ACBP promoters

• Relate to information about stress-responsive rice ACBPs

Functional complementation:

• Express Ricinus communis ACBPs in Arabidopsis or rice acbp mutants

• Test for restoration of stress tolerance

• Similar to expressing OsACBP6 in the Arabidopsis pxa1 mutant, which restored responses related to jasmonic acid accumulation