Recombinant Acaryochloris marina Circadian clock protein kaiA (kaiA)

What is the domain architecture of Acaryochloris marina KaiA compared to other cyanobacterial KaiA proteins?

Acaryochloris marina KaiA belongs to the double-domain KaiA (ddKaiA) subfamily, characterized by a structure approximately 300 amino acids in length. The domain architecture of KaiA proteins can be classified into two main subfamilies:

• Double-domain KaiA (ddKaiA): Found in Oscillatoriophycideae, Synechococcales, Pleurocapsales, and Spirulinales, including A. marina

• Single-domain KaiA (sdKaiA): Found in Chroococcidiopsidales and Nostocales, with variable lengths ranging from 89 to 202 amino acid residues

The C-terminal domain of KaiA is more evolutionarily conserved (dN = 0.30 ± 0.03, π = 0.36 ± 0.00) than the N-terminal domain (dN = 0.88 ± 0.06, π = 0.52 ± 0.00), likely due to its critical role in binding KaiB and KaiC proteins .

How do I express and purify recombinant A. marina KaiA protein for in vitro studies?

Recombinant A. marina KaiA can be expressed and purified using the following methodology:

• Cloning: Clone the A. marina kaiA gene into an expression vector such as pET-28b with a hexahistidine (His) tag

• Expression: Transform into an E. coli expression strain (BL21 or similar) and induce with IPTG

• Purification:

  • Lyse cells in buffer containing appropriate protease inhibitors

  • Perform initial purification using Ni-NTA affinity chromatography

  • Further purify using size-exclusion chromatography to obtain homogeneous protein preparations

  • Verify purity by SDS-PAGE and protein activity through functional assays

The stability of recombinant KaiA can be enhanced by including 10% glycerol in storage buffers and maintaining aliquots at -80°C until use.

How does A. marina KaiA interact with KaiC to regulate phosphorylation?

A. marina KaiA regulates KaiC through transient interactions that stimulate KaiC autokinase activity. Based on studies of cyanobacterial KaiA proteins:

• Binding dynamics: KaiA transiently interacts with KaiC, with high-speed atomic force microscopy (HS-AFM) showing binding/unbinding events occurring on sub-second timescales

• Regulatory mechanism: KaiA enhances both the autophosphorylation and ATPase activities of KaiC

• Feedback regulation: As KaiC becomes more phosphorylated, KaiA's affinity for KaiC progressively weakens, creating a feedback mechanism that is essential for the oscillatory nature of the system

• Binding sites: KaiA primarily interacts with the C-terminal region of KaiC, with conserved residues M241, D242, E251, L265, and D267 (100% conserved across cyanobacteria) likely playing critical roles in this interaction

This dynamic interaction pattern integrates high-frequency binding events to establish the longer-term circadian period.

How do I reconstitute a functional circadian oscillator using recombinant A. marina KaiA in vitro?

To reconstitute an in vitro circadian oscillator using A. marina KaiA:

• Protein components: Purify recombinant KaiA, KaiB, and KaiC proteins (either all from A. marina or in heterologous combinations)

• Reaction setup:

  • Mix the purified Kai proteins in reaction buffer containing ATP

  • Typical ratios: KaiA:KaiB:KaiC = 1:1:2 or 1:3:6 (adjust based on specific experimental goals)

  • Incubate at 30°C under constant conditions

• Monitoring oscillations:

  • Track KaiC phosphorylation states using SDS-PAGE to visualize mobility shifts

  • Sample at regular intervals (e.g., every 4 hours) over 72+ hours

  • Quantify phosphorylation levels by densitometry analysis

Research has shown that LbKaiC1 can oscillate with KaiA and KaiB from Synechococcus elongatus, suggesting some flexibility in reconstituting heterologous systems . A. marina KaiA may similarly function with KaiB and KaiC from related species, though with potentially altered period lengths (approximately 30 hours observed in some heterologous combinations compared to the typical 24-hour period) .

How has the A. marina KaiA protein evolved compared to other cyanobacterial KaiA proteins?

The evolutionary history of A. marina KaiA reflects both conservation and adaptation:

• Ancient origin: Contrary to previous reports, kaiA has an ancient origin and is as old as cyanobacteria themselves, with homologs present in nearly all analyzed cyanobacteria except Gloeobacter

• Domain architecture evolution: Major structural modifications in kaiA genes (duplications, acquisition, and loss of domains) appear to have been driven by global environmental changes across different geological periods

• Evolutionary timeline: Key evolutionary events in kaiA evolution include:

Table: Estimated timing of major evolutionary events in kaiA evolution

• Selection pressures: None of the applied evolutionary analysis methods detected positive selection in kaiA genes, suggesting the protein evolves primarily under purifying selection

What adaptations does A. marina's circadian system show in relation to its unique chlorophyll d-based photosynthesis?

A. marina's circadian system shows several adaptations related to its unique photobiology:

• Niche adaptation: A. marina uses chlorophyll d as its primary photosynthetic pigment, allowing it to utilize far-red light for photosynthesis . Its circadian system likely coevolved with this unique photosynthetic adaptation to optimize light harvesting in specific ecological niches.

• Light sensing integration: The circadian system in A. marina has evolved alongside specialized photoreceptors (cyanobacteriochromes) that detect various wavelengths from ultraviolet to far-red , potentially providing direct input to the KaiABC oscillator.

• Strain diversity: Different A. marina strains show variations in gene content related to light harvesting and regulation. For example:

  • Strain MBIC11017 possesses horizontally acquired phycocyanin genes

  • Other strains like MBIC10699 lack these genes

  • Strain LARK001 exhibits minimal chlorophyll d/a ratio changes in response to different light wavelengths

• Genomic expansion: A. marina has one of the largest bacterial genomes sequenced (8.3 million base pairs) , with extensive gene duplication and horizontal gene transfer potentially allowing for enhanced adaptability of its circadian system to specialized light environments.

How do I determine the structure of A. marina KaiA within the complete KaiABC complex?

Determining the structure of A. marina KaiA within the complete KaiABC complex requires an integrative structural biology approach:

This integrative approach has revealed that the N-terminal domains of KaiA show large fluctuations but preferentially position to mask the hydrophobic surface of the KaiA C-terminal domains, hindering additional KaiA-KaiC interactions .

What are the key conserved residues in A. marina KaiA critical for interaction with KaiC?

Based on evolutionary conservation analysis across cyanobacterial KaiA proteins, several key residues likely play critical roles in A. marina KaiA function:

• 100% conserved residues: Five sites are 100% conserved across all cyanobacteria, including truncated KaiA homologs:

  • M241, D242, E251, L265, and D267

  • These residues are likely essential for the fundamental function of KaiA in all species

• Functional significance of conserved sites:

  • Several conserved residues are involved in maintaining KaiA homodimer structure

  • Others are critical for binding the KaiC protein

• Experimental verification approaches:

  • Site-directed mutagenesis of these conserved residues

  • In vitro phosphorylation assays to measure effects on KaiC regulation

  • Protein-protein interaction assays (e.g., isothermal titration calorimetry, surface plasmon resonance) to quantify binding affinity changes

Mutations in these key residues would be expected to significantly disrupt KaiA function and potentially abolish circadian rhythmicity.

How can recombinant A. marina KaiA be used to study circadian rhythm resilience mechanisms?

Recombinant A. marina KaiA provides a valuable tool for investigating circadian rhythm resilience:

• Stoichiometry variation studies:

  • KaiA's differential affinity phenomenon (weakening binding as KaiC becomes phosphorylated) broadens the range of Kai protein stoichiometries that allow rhythmicity

  • This explains how oscillations remain resilient despite variations in protein levels

  • Experimental approach: Reconstitute in vitro oscillators with varying KaiA:KaiB:KaiC ratios and measure rhythm persistence

• Temperature compensation:

  • Test oscillator function across temperature ranges (20-35°C)

  • Compare A. marina KaiA with other cyanobacterial KaiA proteins to identify structural features contributing to temperature compensation

• Noise resistance:

  • The sub-second binding/unbinding dynamics of KaiA contribute to noise resistance on the 24-hour scale

  • Reconstituted systems can be subjected to controlled perturbations (ATP level fluctuations, temperature pulses) to measure resilience

How does heterologous expression of A. marina KaiA affect circadian rhythms in other cyanobacteria?

Heterologous expression of A. marina KaiA in other cyanobacteria can provide insights into circadian clock plasticity and compatibility:

• Cross-species compatibility:

  • Research has shown that heterologous combinations using KaiA and KaiB from one species can drive oscillations of KaiC from another species, though often with altered period lengths

  • A. marina KaiA could potentially drive oscillations in species lacking functional KaiA or with truncated versions

• Experimental approach:

  • Clone A. marina kaiA under control of an inducible promoter

  • Transform into recipient cyanobacteria (e.g., Synechococcus elongatus PCC 7942)

  • Monitor circadian rhythms using reporter strains (e.g., luciferase reporters)

  • Compare with native rhythms for period, phase, and amplitude differences

• Expected outcomes:

  • Period length alterations (potentially longer periods, as seen in heterologous systems showing ~30h periods versus typical 24h cycles)

  • Potential changes in temperature compensation properties

  • Altered entrainment characteristics to light/dark cycles

• Application potential:

  • Engineering cyanobacteria with modified circadian properties for optimized bioproduction cycles

  • Restoring circadian function in species with naturally occurring kaiA mutations or deletions

What are common challenges in expressing soluble recombinant A. marina KaiA and how can they be overcome?

Common challenges and solutions for recombinant A. marina KaiA expression:

• Insolubility issues:

  • Challenge: KaiA may form inclusion bodies during high-level expression

  • Solutions:

    • Lower induction temperature (16-18°C)

    • Reduce IPTG concentration (0.1-0.2 mM)

    • Co-express with molecular chaperones (GroEL/GroES)

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

• Protein stability problems:

  • Challenge: Purified KaiA may be unstable or aggregate during storage

  • Solutions:

    • Include stabilizing agents (10% glycerol, 1-5 mM DTT)

    • Store at higher concentrations (>1 mg/ml)

    • Avoid freeze-thaw cycles by preparing single-use aliquots

• Functional heterogeneity:

  • Challenge: Variable activity in different preparations

  • Solutions:

    • Implement rigorous quality control tests (SEC analysis, thermal shift assays)

    • Verify functional activity through KaiC phosphorylation assays

    • Ensure complete removal of contaminating proteases

How do I optimize in vitro reconstitution conditions specifically for A. marina KaiABC oscillator systems?

Optimizing in vitro reconstitution conditions for A. marina KaiABC systems:

• Buffer optimization:

  • Test various buffer compositions (HEPES, Tris, phosphate) at pH 7.0-8.0

  • Optimize salt concentrations (typically 150-200 mM KCl or NaCl)

  • Determine optimal ATP concentration (typical range: 1-5 mM)

  • Include magnesium (5-10 mM MgCl₂) as a cofactor for ATPase activity

• Protein ratio determination:

  • Systematically vary KaiA:KaiB:KaiC ratios

  • Common starting ratios: 1:1:4, 1:3:6, or 2:2:6

  • Measure oscillation quality (amplitude, period stability) for each condition

  • The heterologous combination of A. marina KaiC with KaiA and KaiB from other species may require adjusted ratios

• Temperature effects:

  • A. marina optimal growth temperature is typically lower than model species like Synechococcus

  • Test oscillations at 25°C, 30°C, and 35°C

  • The period length of oscillations may vary with temperature (approximately 30h periods have been observed in some heterologous systems)

• Sample handling considerations:

  • Implement careful temperature control throughout the experiment

  • Use low-protein-binding tubes

  • Consider adding BSA (0.1 mg/ml) to prevent protein adsorption to vessel walls

  • For extended experiments (>3 days), seal reaction vessels to prevent evaporation

These optimized conditions will facilitate robust and reproducible circadian oscillations in the reconstituted A. marina KaiABC system.