Recombinant Arabidopsis thaliana Photosystem I reaction center subunit psaK, chloroplastic (PSAK)

What is the functional significance of PSAK in Arabidopsis thaliana?

PSAK serves as a critical peripheral subunit within Photosystem I (PSI), functioning primarily as a connector between the light-harvesting complex I (LHCI) and the PSI core complex. Studies have demonstrated that PSAK-deficient mutants exhibit altered energy transfer patterns within the PSI complex, though they typically maintain photoautotrophic growth capacity under standard laboratory conditions. The protein contains transmembrane domains that anchor it within the thylakoid membrane, with specific regions extending into the stromal side to facilitate protein-protein interactions with neighboring subunits. Unlike the PSII system where D1 C-terminal processing is essential for assembly of functional complexes, PSAK insertion does not require post-translational modification for integration into PSI .

What expression systems work best for recombinant PSAK production?

For optimal expression of recombinant PSAK:

• Bacterial expression: Use E. coli BL21(DE3) or Rosetta strains with pET vectors containing PSAK coding sequence (without transit peptide). Add a solubility tag (MBP, SUMO, or TrxA) to enhance protein folding and reduce inclusion body formation.

• Expression conditions: Induce with 0.2-0.5 mM IPTG at 16-18°C for 16-18 hours in Terrific Broth supplemented with 0.5% glucose and 1% glycerol.

• Extraction protocol: Solubilize membrane fractions with mild detergents (0.5-1% n-dodecyl-β-D-maltoside) at 4°C for 1 hour followed by ultracentrifugation.

• Purification strategy: Apply two-step chromatography (affinity followed by size exclusion) maintaining detergent concentration above critical micelle concentration throughout.

Yields typically range from 0.5-2 mg/L culture, with protein activity highly dependent on maintaining appropriate membrane-mimetic environments during purification.

How can researchers verify successful integration of recombinant PSAK into thylakoid membranes?

Verification of PSAK integration requires multiple complementary approaches:

• Blue Native PAGE analysis: Extract thylakoid membranes and separate native protein complexes to detect PSAK within PSI-LHCI supercomplexes. Look for characteristic bands at approximately 550-650 kDa representing intact PSI-LHCI complexes.

• Immunolocalization: Use anti-PSAK antibodies for Western blot detection in membrane fractions or immunogold labeling for electron microscopy visualization within thylakoid structures.

• Fluorescence microscopy: Create GFP-PSAK fusion constructs for in vivo localization studies in protoplasts or stable transgenic lines.

• Functional reconstitution: Measure P700 oxidation kinetics and 77K fluorescence emission spectra to assess energy transfer efficiency as indicators of proper PSAK integration.

• Protease protection assays: Determine membrane topology by treating intact thylakoids with proteases and analyzing PSAK fragmentation patterns.

These combined approaches provide complementary evidence for both localization and functional integration .

What methods are most effective for studying protein-protein interactions between PSAK and other PSI components?

For investigating PSAK interactions with other PSI components:

• Crosslinking mass spectrometry (XL-MS): Use chemical crosslinkers (DSS, BS3, or EDC) to stabilize native interactions followed by LC-MS/MS analysis. This reveals direct interaction partners and specific contact points between PSAK and neighboring proteins.

• Co-immunoprecipitation with detergent optimization: Solubilize thylakoid membranes with digitonin (0.5%) or n-dodecyl-β-D-maltoside (1%) and perform pull-down experiments using anti-PSAK antibodies. Analyze co-precipitated proteins using LC-MS/MS.

• Bimolecular Fluorescence Complementation (BiFC): Create split-YFP fusion constructs of PSAK and potential interaction partners. Express in Arabidopsis protoplasts and visualize interaction-dependent fluorescence reconstitution via confocal microscopy.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Compare deuterium uptake patterns of isolated PSI complexes with and without PSAK to identify protected regions indicative of interaction interfaces.

• Surface plasmon resonance (SPR): Immobilize purified recombinant PSAK and measure binding kinetics with other PSI subunits to determine association/dissociation constants.

Each method reveals different aspects of the interaction landscape, requiring integration of multiple approaches to establish a comprehensive interaction network .

How do mutations in PSAK affect PSI complex assembly and function?

The effects of PSAK mutations can be characterized using the following methodological approaches:

• Site-directed mutagenesis strategy: Target conserved residues in transmembrane domains and stromal-facing regions. Create alanine scanning mutations across charged/polar residues at predicted protein-protein interfaces.

• Complementation analysis: Transform PSAK-deficient plants with mutated variants under control of the native promoter. Evaluate restoration of wild-type phenotypes under various light conditions.

• Complex assembly assessment: Use Blue Native PAGE and sucrose gradient ultracentrifugation to analyze PSI-LHCI supercomplex formation. Quantify the proportion of fully assembled complexes versus subcomplexes or free proteins.

• Functional measurements:

  • Photosynthetic electron transport rates using Clark-type oxygen electrode

  • P700 oxidation kinetics to evaluate PSI reaction center function

  • 77K chlorophyll fluorescence emission spectra to assess energy transfer efficiency

  • Non-photochemical quenching parameters under fluctuating light conditions

• Structural analysis: Perform cryo-EM analysis of isolated complexes to determine structural perturbations caused by specific mutations.

What are the optimal conditions for reconstituting PSAK into artificial membrane systems?

For successful reconstitution of recombinant PSAK:

• Liposome preparation:

  • Use a lipid mixture mimicking thylakoid composition (MGDG:DGDG:SQDG:PG at 40:30:15:15 mol%)

  • Form liposomes via extrusion through 100 nm polycarbonate membranes

  • Maintain pH 7.5-8.0 and include 100-150 mM NaCl to stabilize membrane structure

• Protein incorporation methodology:

  • Direct incorporation: Gradually remove detergent using Bio-Beads SM-2 or dialysis against detergent-free buffer

  • Mechanical reconstitution: Mix protein-detergent micelles with preformed liposomes followed by freeze-thaw cycles

  • Optimize protein:lipid ratio (typically 1:100 to 1:500 w/w)

• Verification techniques:

  • Dynamic light scattering to confirm proteoliposome size distribution

  • Sucrose density gradient centrifugation to separate proteoliposomes from empty liposomes

  • Freeze-fracture electron microscopy to visualize protein distribution

  • Fluorescence recovery after photobleaching (FRAP) to assess protein mobility

• Functional assessment:

  • Measure energy transfer using fluorescence spectroscopy

  • Probe conformational changes via circular dichroism

  • Assess interaction with other PSI components in the artificial system

Successful reconstitution provides a platform for studying PSAK function in a controlled environment without complications from other chloroplast components .

How can CRISPR-Cas9 be optimized for precise editing of the PSAK gene?

For CRISPR-Cas9 editing of the PSAK gene in Arabidopsis:

• gRNA design parameters:

  • Select target sites with minimal off-target effects using CRISPOR or CHOPCHOP tools

  • Ensure high on-target activity score (>50)

  • Target early exons to maximize disruption probability

  • Verify target uniqueness with whole-genome BLAST analysis

  • Design at least three independent gRNAs targeting different regions

• Delivery optimization:

  • Use egg cell-specific promoters (EC1.2) for Cas9 expression to enhance germline editing

  • Employ Arabidopsis codon-optimized Cas9 with nuclear localization signals

  • Deliver via Agrobacterium-mediated floral dip transformation

  • Screen T1 transformants for editing efficiency

• Repair template strategy:

  • For precise edits, design single-stranded oligodeoxynucleotide (ssODN) repair templates

  • Include 30-50 bp homology arms flanking the desired mutation

  • Introduce silent mutations in the PAM site to prevent re-cutting

  • Add restriction sites for simplified screening

• Screening protocol:

  • Develop PCR-based genotyping assays for detecting edits

  • Implement Sanger sequencing to confirm mutations

  • Verify phenotypic changes with chlorophyll fluorescence analysis

This approach allows for precise engineering of specific amino acid substitutions to investigate structure-function relationships in PSAK .

What are the most reliable methods for studying PSAK phosphorylation?

To investigate PSAK phosphorylation:

• Phosphorylation site identification:

  • Enrich phosphopeptides using TiO₂ or immobilized metal affinity chromatography

  • Analyze via LC-MS/MS with neutral loss scanning for phosphate groups

  • Compare phosphorylation patterns under different light conditions and stress treatments

  • Validate sites using phospho-specific antibodies

• Functional significance assessment:

  • Generate phosphomimetic (S/T→D/E) and phospho-null (S/T→A) mutants

  • Express in psak knockout backgrounds

  • Compare PSI assembly, stability, and function across variants

  • Measure electron transport rates under different light intensities

• Kinase identification:

  • Perform in vitro kinase assays with thylakoid extracts

  • Use inhibitor profiling to narrow down kinase families

  • Conduct Y2H screens with known chloroplast kinases

  • Validate via co-immunoprecipitation and in vitro phosphorylation

• Physiological relevance determination:

  Condition	Phosphorylation Level	Functional Impact
  High light	Increased (2-3×)	Enhanced LHCI dissociation
  Cold stress	Increased (1.5-2×)	Altered energy distribution
  State transitions	Dynamic changes	Regulates PSI-LHCI association
  Developmental stages	Age-dependent pattern	Affects complex stability

Phosphorylation typically occurs at the stromal-exposed regions of PSAK and may regulate its interaction with LHCI components and other PSI subunits .

How can researchers optimize isotope labeling of recombinant PSAK for structural studies?

For successful isotope labeling of PSAK:

• Expression system selection:

  • Use E. coli BL21(DE3) with T7 promoter-based vectors

  • Consider cell-free expression systems for difficult constructs

• Media composition for different labeling schemes:

  Labeling Type	Base Media	Key Components	Expression Temperature	Yield (mg/L)
  ¹⁵N	M9 minimal	¹⁵NH₄Cl (1g/L)	18°C	0.5-1.0
  ¹³C	M9 minimal	¹³C-glucose (2g/L)	18°C	0.4-0.8
  ¹³C,¹⁵N	M9 minimal	¹⁵NH₄Cl + ¹³C-glucose	18°C	0.3-0.7
  Deuteration	D₂O-M9	D₂O (70-99%)	18°C	0.2-0.5
  Selective labeling	M9 + amino acids	Labeled amino acids	18°C	0.7-1.2

• Solubilization optimization:

  • Screen detergent panel (DDM, DPC, LDAO)

  • Test amphipols (A8-35) for NMR studies

  • Consider nanodiscs with MSP1D1 and thylakoid-mimicking lipids

• Purification protocol:

  • Implement two-step affinity chromatography

  • Verify isotope incorporation by mass spectrometry

  • Confirm proper folding via circular dichroism

• NMR sample preparation:

  • Concentrate to 0.3-0.5 mM in deuterated buffer

  • Optimize temperature (25-40°C) and pH (6.5-7.5)

  • Use 5mm Shigemi tubes to minimize sample volume

This approach enables structural studies of PSAK in membrane-mimetic environments while maintaining its native fold .

What techniques are most effective for analyzing the dynamics of PSAK turnover in vivo?

To study PSAK protein turnover dynamics:

• Pulse-chase analysis:

  • Label proteins with ³⁵S-methionine (30-minute pulse)

  • Chase with excess unlabeled methionine

  • Isolate thylakoids at various timepoints (0-24 hours)

  • Immunoprecipitate PSAK and quantify remaining radioactivity

  • Calculate half-life under different environmental conditions

• Fluorescent timer fusion proteins:

  • Create PSAK fusion with fluorescent timer proteins (e.g., DsRed-E5)

  • Transform psak knockout plants

  • Measure changes in fluorescence spectrum over time

  • Calculate protein age based on spectral shifts

• Conditional expression systems:

  • Generate inducible PSAK expression lines

  • Induce expression briefly, then turn off

  • Track protein levels via immunoblotting

  • Fit degradation curves to determine decay constants

• Inhibitor studies:

  • Apply specific protease inhibitors (FtsH, Clp, DEG proteases)

  • Monitor PSAK stability following inhibitor treatment

  • Identify primary degradation pathways

• Turnover rates under different conditions:

  Condition	Half-life (hours)	Major Degradation Pathway
  Normal light	18-24	Gradual FtsH-mediated
  High light	4-6	Accelerated FtsH + Clp
  Dark adaptation	30-36	Minimal turnover
  Heat stress	2-3	Rapid Clp-dependent
  Cold stress	12-16	Intermediate rate

These approaches reveal that PSAK turnover is regulated by light conditions and stress factors, with specific proteolytic pathways activated under different environmental challenges .

How does PSAK structure and function compare between Arabidopsis and other photosynthetic organisms?

Comparative analysis of PSAK across species reveals:

• Structural conservation patterns:

  Organism	PSAK Size (aa)	TMD Number	Conservation Level	Unique Features
  Arabidopsis thaliana	139	2	Reference	Extended stromal loop
  Chlamydomonas reinhardtii	121	2	65% identity	Shorter N-terminus
  Synechocystis sp. PCC 6803	86	1	42% identity	Single transmembrane helix
  Spinacia oleracea	142	2	89% identity	Similar to Arabidopsis
  Pisum sativum	140	2	88% identity	Similar to Arabidopsis

• Functional divergence:

  • Cyanobacterial PSAK (PsaK) contains a single transmembrane domain and lacks the extended stromal loop present in plant versions

  • Green algal PSAK shows intermediate complexity between cyanobacterial and land plant forms

  • Land plant PSAKs contain conserved phosphorylation sites absent in cyanobacterial homologs

  • Role in LHCI binding appears specialized in land plants compared to cyanobacteria

• Evolutionary significance:

  • PSAK evolution correlates with increasing complexity of light-harvesting antenna systems

  • Acquisition of second transmembrane domain coincides with evolution of LHCI proteins

  • Phosphorylation sites emerged as regulatory mechanisms for dynamic antenna adjustment

  • Conserved sequence motifs identify functional residues for protein-protein interactions

This evolutionary comparison helps identify critical residues for targeted mutagenesis and functional studies .

How can heterologous expression systems be optimized for PSAK from different species?

Optimization strategies for heterologous PSAK expression:

• Expression host selection criteria:

  Host System	Advantages	Disadvantages	Best For
  E. coli	Rapid growth, high yield	Lacks chloroplast machinery	Basic structural studies
  Chloroplast-containing algae	Native-like processing	Lower yields, complex genetics	Functional studies
  Plant cell cultures	Native post-translational modifications	Slow growth, expensive	Complex formation studies
  Cell-free systems	Control over redox environment	Higher cost, limited scale	Difficult constructs

• Codon optimization parameters:

  • Adapt to expression host codon usage bias

  • Eliminate rare codons and potential RNA secondary structures

  • Optimize GC content based on expression system

  • Remove cryptic splice sites for eukaryotic expression

• Fusion protein design:

  • N-terminal fusions preferred (TEV-cleavable MBP, SUMO, TrxA)

  • Consider GFP fusion for folding assessment

  • Optimize linker length (15-20 amino acids) for membrane proteins

• Expression condition optimization:

  • Temperature gradient screening (16-30°C)

  • Inducer concentration titration

  • Media supplementation (glycerol, sorbitol)

  • Co-expression with chaperones (GroEL/ES, DnaK/J)

• Species-specific considerations:

  • Cyanobacterial PSAK: Express without fusion partners due to smaller size

  • Green algal PSAK: Requires optimization of membrane insertion

  • Land plant PSAK: Benefits from lipid supplementation during expression

These approaches enable comparative studies of PSAK structure-function relationships across evolutionary lineages .