Recombinant Escherichia coli O81 Acyl carrier protein (acpP)

What is the biological role of Acyl Carrier Protein in E. coli fatty acid synthesis?

Acyl Carrier Protein (ACP) serves as the central shuttle that carries growing fatty acid intermediates between the various enzymes in the type II fatty acid synthesis (FAS) pathway. In E. coli, ACP (encoded by the acpP gene) interacts with at least five different protein partners during each cycle of fatty acid chain elongation . The protein functions by covalently binding acyl intermediates through a thioester linkage to its 4'-phosphopantetheine prosthetic group, allowing it to transport these intermediates between enzymatic active sites while protecting them from cytosolic degradation .

During fatty acid synthesis, ACP undergoes a series of sequential interactions:

• It accepts malonyl groups from malonyl-CoA via FabD (malonyl-CoA:ACP transacylase)

• It participates in condensation reactions catalyzed by FabH (initiating reactions) or FabB/FabF (elongation)

• It carries 3-ketoacyl intermediates to FabG (3-oxoacyl-ACP reductase)

• It transports 3-hydroxyacyl intermediates to FabZ (dehydratase)

• It delivers trans-2-enoyl intermediates to FabI (enoyl-ACP reductase)

These protein-protein interactions are crucial for metabolic efficiency and regulation of fatty acid synthesis.

How is recombinant E. coli acpP properly activated for functional studies? (Basic)

The biological activity of acpP requires post-translational modification through the attachment of a 4'-phosphopantetheine group from Coenzyme A to a conserved serine residue (Ser36 in E. coli acpP). This modification is catalyzed by the enzyme AcpS (holo-ACP synthase) .

Methodological approach:

• Express recombinant acpP in E. coli BL21(DE3) using an appropriate expression vector (e.g., pET28b)

• Co-express with AcpS or perform in vitro modification:

  • In vitro conversion: Incubate purified apo-ACP with CoA and AcpS in buffer containing Mg²⁺

  • Verify modification status using conformationally sensitive gel electrophoresis or mass spectrometry

• Purify using nickel chelate chromatography if His-tagged

• Confirm holo-ACP formation through:

  • Mass spectrometry (mass increase of 340 Da)

  • Functional assays (ability to accept acyl groups)

When expressed in E. coli, approximately 50-80% of recombinant acpP is typically converted to holo-form due to endogenous AcpS activity, but complete conversion may require additional in vitro treatment .

What expression systems and purification methods yield functionally active recombinant acpP? (Basic)

Expression systems:

Purification methodology:

• Affinity chromatography approach:

  • N-terminal His₆-tagged constructs in pET vectors show high expression

  • Purify using nickel chelate chromatography with imidazole gradient elution

  • Consider tag removal if it interferes with function

• Non-tagged purification approach:

  • Heat treatment (65°C for 5 min) exploiting acpP's thermal stability

  • Anion exchange chromatography (ACP is highly acidic, pI ~4.1)

  • Hydrophobic interaction chromatography

  • Size exclusion as final polishing step

• Quality control:

  • Determine purity by SDS-PAGE (acpP runs at ~10 kDa despite 8.5 kDa mass)

  • Verify holo-form using conformationally sensitive gel electrophoresis

  • Test functionality in acylation assays with acyl-ACP synthetase

Typical yields range from 15-30 mg of purified recombinant acpP per liter of culture medium with >90% purity achievable using these methods .

How can I verify the modification status and functionality of recombinant acpP? (Advanced)

Determination of modification status and functionality requires analytical and functional approaches:

Analytical methods:

• Mass spectrometry analysis:

  • ESI-MS or MALDI-TOF MS to distinguish apo- from holo-acpP

  • Expected mass difference: +340 Da for holo-form

  • Acylated forms show additional mass increases based on acyl chain length

• Conformationally sensitive gel electrophoresis:

  • Urea-PAGE (1M urea) separates apo- and holo-forms due to conformational differences

  • Holo-form typically migrates faster than apo-form

  • Acylated forms show even greater mobility shifts

Functional assays:

• Acylation assay:

  • Incubate acpP with V. harveyi AasS, ATP, and fatty acid (e.g., octanoic acid)

  • Only holo-acpP can be acylated

  • Monitor by conformationally sensitive gel electrophoresis or mass spectrometry

• In vitro fatty acid synthesis reconstitution:

  • Combine purified acpP with FabD, FabH, FabG, FabZ, and FabI

  • Add malonyl-CoA and acetyl-CoA substrates

  • Monitor acyl-ACP formation by gel electrophoresis or LC-MS

  • Complete pathway function indicates properly modified and functional acpP

How can recombinant acpP be used to study protein-protein interactions in fatty acid synthesis? (Advanced)

Recombinant acpP serves as a valuable tool for investigating protein-protein interactions within the fatty acid synthesis pathway:

Methodological approaches:

• Crosslinking studies:

  • Use phosphopantetheinamide analogs to trap ACP-enzyme complexes

  • Apply mechanistically-compatible crosslinkers that covalently capture interaction states

  • Analyze complexes by SDS-PAGE, Western blotting, and mass spectrometry

• NMR chemical shift perturbation analysis:

  • Prepare ¹⁵N-labeled recombinant acpP

  • Record HSQC spectra in the presence and absence of partner enzymes

  • Map interaction interfaces by analyzing chemical shift changes

  • Research has shown that FabG-acpP interactions occur along helix α2 and the adjacent loop-2 region of acpP

• Alanine scanning mutagenesis:

  • Systematically replace surface residues of acpP with alanine

  • Assess impact on binding to partner enzymes using enzymatic assays

  • Key electronegative/hydrophobic residues along helix α2 are critical for recognition by partner enzymes

• Tandem affinity purification:

  • Express tagged acpP in E. coli to capture interaction partners

  • Identify novel protein associations by mass spectrometry

  • This approach has revealed unexpected interactions beyond fatty acid synthesis pathway components

Recent studies identified that mutations in FabG at positions Arg129 and Arg172 significantly reduce binding to acpP, indicating that electropositive residues embedded in hydrophobic patches on enzyme surfaces are critical for acpP recognition .

What are the essential controls and considerations for in vitro fatty acid synthesis assays using recombinant acpP? (Advanced)

In vitro fatty acid synthesis reconstitution assays require careful experimental design:

Essential components and controls:

• Required components:

  • Purified holo-acpP (>95% pure)

  • Pathway enzymes: FabD, FabH, FabG, FabZ, FabI

  • Substrates: malonyl-CoA, acetyl-CoA

  • Cofactors: NADPH, NADH

  • Buffer: typically 0.1 M sodium phosphate (pH 7.0)

• Critical controls:

  • Enzyme-free control to assess non-enzymatic reactions

  • Individual enzyme omission controls to verify each step

  • Apo-acpP control (non-functional) to confirm holo-form requirement

  • Heterologous acpP (e.g., spinach ACP-I) to assess specificity

Analytical methods:

• Conformationally sensitive gel electrophoresis:

  • Separates different acyl-ACP intermediates

  • Allows time-course monitoring of fatty acid synthesis

  • Observe distinct bands for each acyl-chain length

• LC-MS analysis:

  • Provides quantitative data on acyl-ACP species

  • Allows identification of reaction intermediates

  • Can detect unexpected side products

Data interpretation:

Research has shown that long-chain acyl-ACPs can inhibit acetyl-CoA carboxylase, demonstrating the importance of monitoring product inhibition in these assays .

How can site-directed mutagenesis of acpP be used to investigate protein-protein interactions in the fatty acid synthesis pathway? (Advanced)

Site-directed mutagenesis of acpP provides valuable insights into the molecular determinants of protein-protein interactions:

Methodological approach:

• Identifying target residues:

  • Focus on helix α2 residues (amino acids 35-50)

  • Target the conserved DSL motif surrounding the 4'-phosphopantetheine attachment site

  • Consider loop regions that undergo conformational changes during interactions

  • Use structural information and sequence conservation analysis to guide selection

• Mutation strategies:

  • Charge reversal mutations (e.g., Asp→Arg) to disrupt electrostatic interactions

  • Conservative substitutions to assess specificity requirements

  • Alanine scanning to identify critical interaction residues

  • Introduction of non-native residues to probe binding pocket dimensions

• Functional assessment:

  • Express and purify mutant proteins

  • Conduct in vitro binding assays with partner enzymes

  • Measure enzymatic activity in reconstituted systems

  • Perform structural studies (e.g., NMR, X-ray crystallography) of mutant-enzyme complexes

Research findings:

Studies have demonstrated that the electronegative and hydrophobic residues along helix α2 of acpP are crucial for recognition by partner enzymes. The most conserved residues in this region are particularly important for interactions with FabG and other fatty acid synthesis enzymes .

Specific mutations in E. coli acpP that significantly affect function include:

These findings highlight the importance of both electrostatic and hydrophobic interactions in acpP-enzyme recognition.

What approaches can be used to study the dynamics of acpP during fatty acid synthesis? (Advanced)

Understanding the dynamic behavior of acpP during fatty acid synthesis requires specialized techniques:

Methodological approaches:

• NMR dynamics studies:

  • Express ¹⁵N/¹³C-labeled acpP

  • Perform relaxation measurements to assess backbone dynamics

  • Use CPMG and relaxation dispersion to detect conformational exchange

  • Apply NOESY experiments to monitor acyl chain interactions with ACP hydrophobic core

• FRET-based approaches:

  • Introduce fluorescent labels at strategic positions on acpP

  • Express fluorescently labeled partner enzymes

  • Monitor real-time interactions using single-molecule FRET

  • Track pathway progression through sequential enzyme interactions

• Molecular dynamics simulations:

  • Generate computational models of acpP in various acylation states

  • Simulate conformational changes during acyl chain growth

  • Model protein-protein docking and interaction dynamics

  • Validate computational predictions experimentally

• Hydrogen-deuterium exchange mass spectrometry:

  • Expose acpP-enzyme complexes to D₂O buffer

  • Quench at various timepoints and analyze by MS

  • Identify regions with altered solvent accessibility

  • Map conformational changes during complex formation

Research has revealed that acpP undergoes significant conformational changes during acylation, with the acyl chain sequestered within a hydrophobic pocket that expands to accommodate longer chains. During interactions with partner enzymes, a process called "chain flipping" occurs, where the acyl chain exits the ACP pocket and enters the enzyme active site .

What experimental design principles should be applied when using recombinant acpP in complex enzymatic assays? (Basic)

When designing experiments with recombinant acpP, several principles ensure reliable and interpretable results:

Experimental design considerations:

• Variable control:

  • Identify independent variables (e.g., enzyme concentrations, substrate levels)

  • Define dependent variables (e.g., product formation, reaction rates)

  • Control confounding variables (e.g., temperature, buffer composition)3

• Sampling approach:

  • Run multiple replicates for each condition (minimum n=3)

  • Include appropriate controls for each experiment

  • Consider time-course sampling to establish reaction kinetics3

• Bias minimization:

  • Perform blinded analysis where possible

  • Randomize sample processing order

  • Include positive and negative controls in each experimental batch3

Methodological implementation:

• Sample preparation:

  • Maintain consistent protein concentrations across experiments

  • Verify enzyme activity before complex assays

  • Prepare fresh substrate solutions to avoid degradation

  • Document lot numbers and storage conditions

• Data collection:

  • Use calibrated instruments with appropriate sensitivity

  • Establish linear range for quantitative measurements

  • Include standard curves for absolute quantification

  • Document all experimental conditions and deviations

Studies have shown that seemingly minor variations in experimental conditions can significantly impact the reproducibility of fatty acid synthesis assays3.

How should acpP interaction data be analyzed and interpreted in the context of fatty acid synthesis research? (Advanced)

Proper analysis of acpP interaction data requires appropriate statistical and analytical approaches:

Data analysis methods:

• Kinetic analysis:

  • Determine binding constants (Kd) from titration experiments

  • Calculate kinetic parameters (Km, kcat) for enzymatic reactions

  • Apply appropriate models (Michaelis-Menten, allosteric, etc.)

  • Use global fitting for complex reaction schemes

• Interaction mapping:

  • Generate heat maps of chemical shift perturbations from NMR data

  • Create surface representation of interaction interfaces

  • Compare experimental data with computational docking models

  • Integrate data from multiple experimental approaches

• Statistical considerations:

  • Apply appropriate statistical tests based on data distribution

  • Calculate confidence intervals for kinetic parameters

  • Use ANOVA for multi-condition comparisons

  • Consider Bayesian approaches for complex datasets

Interpretation framework:

Research findings should be interpreted within the broader context of fatty acid synthesis:

Recent studies have demonstrated that E. coli acpP interactions are highly specific, as evidenced by the inability of spinach ACP-I to inhibit E. coli acetyl-CoA carboxylase despite 44% sequence identity and similar three-dimensional structures .

How can recombinant acpP be utilized in synthetic biology applications? (Advanced)

Recombinant acpP offers numerous applications in synthetic biology and metabolic engineering:

Methodological approaches:

• Engineering fatty acid synthesis pathways:

  • Express recombinant acpP variants with altered specificities

  • Engineer novel enzyme-acpP interactions for non-native products

  • Create chimeric acpP proteins with domains from different organisms

  • Optimize acpP expression levels to balance pathway flux

• Producing novel fatty acid derivatives:

  • Express acpP alongside engineered fatty acid synthesis enzymes

  • Incorporate non-native fatty acid synthesis modules

  • Introduce thioesterases with altered specificities for product release

  • Create pathways for methylated, hydroxylated, or cyclic fatty acids

• acpP as a carrier for polyketide and non-ribosomal peptide synthesis:

  • Utilize acpP's carrier function in heterologous pathways

  • Engineer acpP to interact with polyketide synthases

  • Create domain-swapped carrier proteins with novel functions

  • Express acpP variants optimized for specific synthetic pathways

Research applications:

Recent studies have demonstrated the versatility of acpP in synthetic biology applications. For example, researchers have engineered E. coli to produce phosphatidylcholine (PC) and other methylated phospholipid derivatives by expressing acpP alongside heterologous enzymes or by manipulating endogenous pathways .

The large-scale E. coli genotype-phenotype dataset for antimicrobial resistance research represents another advanced application where acpP function could be investigated in the context of membrane composition and antibiotic resistance mechanisms .

What current technological limitations affect recombinant acpP research and how might they be addressed? (Advanced)

Several technological challenges currently limit recombinant acpP research:

Current limitations and potential solutions:

• Heterogeneity in post-translational modification:

  • Challenge: Variable conversion of apo- to holo-acpP

  • Solution approaches:

    • Co-expression systems with optimized AcpS

    • In vitro conversion followed by separation techniques

    • Development of selective purification methods for holo-form

    • Genetic modifications to enhance modification efficiency

• Difficulties in structural analysis of acpP-enzyme complexes:

  • Challenge: Transient nature of interactions

  • Solution approaches:

    • Crosslinking with mechanistically-compatible linkers

    • Time-resolved structural techniques (TR-SAXS, cryo-EM)

    • Development of stabilized complexes through protein engineering

    • Integrative structural biology combining multiple techniques

• Limitations in monitoring real-time dynamics:

  • Challenge: Capturing sequential interactions in multi-enzyme systems

  • Solution approaches:

    • Advanced single-molecule techniques (FRET, TIRF microscopy)

    • Development of non-disruptive fluorescent tags

    • Microfluidic platforms for controlled reaction environments

    • Computational methods to predict and model dynamic interactions

• Challenges in specificity engineering:

  • Challenge: Designing acpP variants with novel specificities

  • Solution approaches:

    • Machine learning approaches to predict interaction determinants

    • High-throughput screening of acpP variant libraries

    • Directed evolution strategies for acpP engineering

    • Rational design based on comprehensive interaction maps

What are the most common issues encountered when working with recombinant acpP and how can they be resolved? (Basic)

Researchers frequently encounter several challenges when working with recombinant acpP:

Common issues and solutions:

• Low expression yields:

  • Problem: Poor soluble expression of recombinant acpP

  • Solutions:

    • Optimize growth temperature (try 18-25°C)

    • Use specialized E. coli strains (e.g., BL21(DE3)pLysS)

    • Reduce inducer concentration (0.1-0.5 mM IPTG)

    • Add solubility-enhancing fusion tags (e.g., SUMO, MBP)

• Incomplete post-translational modification:

  • Problem: Mixture of apo- and holo-acpP forms

  • Solutions:

    • Co-express with AcpS

    • Perform in vitro modification with purified AcpS and CoA

    • Use conformationally sensitive gel electrophoresis to monitor conversion

    • Employ pH shift during purification to separate forms

• Protein instability:

  • Problem: Degradation during purification or storage

  • Solutions:

    • Include protease inhibitors throughout purification

    • Add reducing agents (DTT or β-mercaptoethanol)

    • Store as smaller aliquots at -80°C

    • Add glycerol (10-50%) to storage buffer

• Activity loss in functional assays:

  • Problem: Purified acpP shows poor activity

  • Solutions:

    • Verify holo-form by mass spectrometry

    • Test activity immediately after purification

    • Optimize buffer conditions (pH, salt concentration)

    • Ensure all pathway components are active individually

Research has shown that E. coli acpP is relatively stable but sensitive to oxidation of the 4'-phosphopantetheine thiol group, which can be prevented by including reducing agents in storage buffers .

How can researchers validate the quality and consistency of recombinant acpP preparations across different batches? (Basic)

Ensuring batch-to-batch consistency is crucial for reliable research results:

Quality control methodology:

• Physical characterization:

  • SDS-PAGE for purity assessment (>95% purity recommended)

  • Size exclusion chromatography to detect aggregation

  • Dynamic light scattering for homogeneity analysis

  • Mass spectrometry for accurate mass determination and modification status

• Functional validation:

  • Standardized acylation assay with V. harveyi AasS

  • Consistent enzyme-to-acpP ratios in activity tests

  • Reference standards from previous validated batches

  • Activity comparison under identical conditions

• Documentation requirements:

  • Detailed expression and purification records

  • Storage conditions and freeze-thaw cycles

  • Standard curves for quantification methods

  • Acceptance criteria for each quality parameter

Standardization approach:

• Create master cell banks of expression strains

• Establish standard operating procedures for expression and purification

• Develop reference standards from well-characterized batches

• Implement statistical process control to monitor trends

Experimental design principles, including proper controls and statistical rigor, should be applied to quality control testing to ensure reliable comparisons between batches3 .