Recombinant Prochlorococcus marinus subsp. pastoris Photosystem II reaction center Psb28 protein (psb28)

What is Psb28 and what is its fundamental role in Photosystem II?

Psb28 is the only cytoplasmic extrinsic protein in PSII that transiently associates with PSII assembly intermediates, particularly the RC47 complex (PSII core complex lacking the CP43 antenna). It serves as a protective factor during PSII biogenesis and repair . While most Psb28 exists in an unassembled state in the membrane fraction, a small portion binds to RC47, where it plays a crucial role in regulating PSII assembly .

Experimentally, this has been demonstrated through protein localization studies using membrane fractionation and isolation of RC47 from strains containing histidine-tagged derivatives of Psb28 via nickel-affinity chromatography . The protein's association with RC47 was further confirmed by preferential isolation of this complex, establishing Psb28's role in PSII biogenesis .

How can researchers effectively express and purify recombinant Psb28 protein?

For successful expression and purification of Psb28, researchers typically employ the following methodology:

• Expression systems: Yeast expression systems have proven effective for producing recombinant Psb28 from Prochlorococcus marinus . For other experimental approaches, E. coli systems have been used successfully to produce recombinant Psb28 for downstream applications such as protein-protein interaction studies and nuclear magnetic resonance (NMR) spectroscopy .

• Purification strategy:

  • Utilize histidine-tagged versions of the protein for affinity purification

  • Implement two-dimensional separation techniques combining blue native (BN)-PAGE in one direction and denaturing PAGE in the second direction for analysis of Psb28 incorporation into complexes

  • For quantitative studies, purified recombinant Psb28 can be used as a standard to estimate cellular abundance via immunoblotting

• Quality control: Evidence from recombinant Psb28 migration patterns on BN gels indicates the protein forms at most dimers but no higher oligomers, which should be verified during purification .

What experimental phenotypes are observed in psb28 deletion mutants?

Deletion of psb28 results in several distinctive phenotypes that provide insight into its function:

Additionally, psb28 mutants from Chlamydomonas exhibit more severe PSII assembly impairments, suggesting organism-specific roles for this protein .

What structural alterations does Psb28 binding induce in the PSII complex?

Psb28 binding induces significant structural changes in PSII that influence both its assembly and protective functions:

• β-hairpin structure formation: Binding of Psb28 induces the formation of an extended β-hairpin structure that incorporates:

  • The central antiparallel β-sheet of Psb28

  • The C-terminus of CP47

  • The D1 D-E loop

• D-E loop conformational changes: Psb28 binding causes large structural changes at the D–E loop regions of D1 and D2, which affects:

  • The environment of the QA/QB binding sites

  • The non-haem iron coordination sphere

• Bicarbonate displacement: Evidence suggests that Psb28 binding may cause displacement of bicarbonate during PSII biogenesis, with the carboxylate side chain of a D2 (PsbD) residue replacing it as a ligand to the non-heme iron . This structural change is proposed to increase the redox potential of QA-- to favor safe charge recombination between P680+- and QA-- , reducing potential photooxidative damage .

• Cytochrome b559 interaction: Cross-linking studies have positioned Psb28 on the cytosolic surface of PSII directly above cytochrome b559 (composed of PsbE and PsbF subunits), in close proximity to the QB site . This location is strategically important for protecting the electron transport chain during assembly.

These structural insights were determined using a combination of cryo-electron microscopy, chemical cross-linking combined with mass spectrometry, and protein-protein docking analyses .

How can researchers experimentally determine Psb28's binding partners and interaction sites?

Multiple complementary techniques have proven effective for identifying Psb28's binding partners:

• Chemical cross-linking with mass spectrometry:

  • Implementation: Use isotope-encoded cross-linking with "mass tags" selection criteria

  • Advantages: Allows capture of transient interactions and provides distance constraints

  • Results: Identified three cross-links between Psb28 and the α- and β-subunits of cytochrome b559

  • Cross-link distances: 15, 10, and 14 Å for the PsbE–Psb28, PsbF–Psb28-K8, and PsbF–Psb28-A2 cross-links, respectively

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Implementation: Chemical shift perturbation (CSP) experiments with recombinant Psb28 and synthetic peptides

  • Results: Identified notable shifts with a chemical shift difference (Δδ) of >1 s.d. located at strands β3 and β4 and the C-terminal region of Psb28

  • Binding parameters: Yielded a dissociation constant (KD) of 53.92 ± 0.41 μM

• Co-immunoprecipitation with LC-MS/MS analysis:

  • Implementation: Immunoprecipitation of tagged Psb28 following in vivo crosslinking

  • Results: In membrane fractions, only D1, D2, CP43, and CP47 were detected in all replicates

  • Quantitative insights: Using intensity-based absolute quantification (IBAQ) normalized to Psb28 revealed that D2 was the most abundant protein in the precipitate, followed by D1, CP47, and CP43

• Blue native gel electrophoresis:

  • Implementation: BN-PAGE analysis of tagged Psb28 migration and co-migration with PSII complexes

  • Results: Demonstrated co-migration with PSII monomers and RC47 complexes

These experimental approaches collectively provide a comprehensive view of Psb28's interaction network within the photosynthetic apparatus.

What is the mechanism by which Psb28 protects PSII during assembly and repair?

Psb28 employs several mechanisms to protect PSII during its biogenesis and repair cycles:

• Modulation of redox chemistry:

  • Psb28 binding causes structural changes that may alter the redox potential of QA, favoring safe charge recombination pathways and reducing singlet oxygen production

  • This modification of the QA/QB environment potentially minimizes photodamage by reducing the production of singlet oxygen from chlorophyll triplet states produced via charge recombination

• Phycobilisome docking prevention:

  • Attachment of Psb28 to the cytoplasmic surface of RC47 prevents docking of the phycobilisome

  • This prevents premature light harvesting by assembly intermediates that lack a fully functional electron transport chain

• QB site protection:

  • Psb28's binding position directly above cytochrome b559 and close to the QB site allows it to block the QB binding site

  • The structure shows that the presence of Psb28 blocks QB binding and alters the structure of the D–E loop of PsbA, in which the QB headgroup is typically found

• PSII repair regulation:

  • Psb28 appears to block assembly of a fraction of newly assembled PSII at the RC47 stage, potentially serving as a checkpoint in the assembly process

  • This regulatory function may be particularly important under stress conditions when PSII damage occurs frequently

These protective mechanisms appear to be especially critical during fluctuating light conditions and under temperature stress, as demonstrated by growth defects of the psb28-1 mutant at 38°C and light intensities of 30 μmol photons m-2 s-1 or higher .

How does Psb28 influence chlorophyll biosynthesis and incorporation into photosystems?

Psb28's role in chlorophyll (Chl) biosynthesis represents one of its most significant functions:

• Specific step in chlorophyll biosynthesis pathway:

  • Psb28 deletion leads to inhibition of chlorophyll biosynthesis specifically at the cyclization step yielding the isocyclic ring E

  • Mutant cells accumulate high levels of magnesium protoporphyrin IX methylester, have decreased levels of protochlorophyllide, and release large quantities of protoporphyrin IX into the medium

• Coordinated synthesis of chlorophyll-binding proteins:

  • Radioactive labeling experiments revealed limitations in the synthesis of both CP47 and the PSI subunits PsaA/PsaB in the absence of Psb28

  • This indicates Psb28's role in coordinating chlorophyll synthesis with the production of chlorophyll-binding proteins

• Differential effects on photosystems:

  • Psb28 deletion affects both PSII and PSI, but with different consequences

  • While PSII repair is accelerated in mutants, PSI content is reduced

  • This suggests Psb28 plays a regulatory role in balancing resources between the two photosystems

The mechanism appears to involve Psb28's interaction with the RC47 intermediate, which could serve as a signaling platform to coordinate chlorophyll synthesis with the availability of chlorophyll-binding proteins, preventing the accumulation of free chlorophyll intermediates that could cause photooxidative damage.

Is Psb28 function conserved across different photosynthetic organisms, and can heterologous Psb28 proteins complement mutant phenotypes?

Psb28 function shows both conservation and divergence across photosynthetic organisms:

• Conservation analysis:

  • Psb28 homologs are found across cyanobacteria, algae, and plants

  • The core function in PSII assembly appears conserved, but with species-specific adaptations

• Complementation studies:

  • Synechocystis Psb28-1 can complement Chlamydomonas psb28 mutants, but with low efficiency

  • Complementation experiments revealed:

    • Seven transformants had Fv/Fm values around or below that of the mutant

    • Only three transformants exhibited improved Fv/Fm values over the psb28 mutant

  • The limited complementation could be due to:

    • Very low abundance due to protein instability

    • Potential cleavage of the HA tag in recombinant constructs

    • Structural differences affecting interaction with photosystem components

• Organism-specific differences:

  • In cyanobacteria like Synechocystis, Psb28 deletion causes subtle phenotypes with accelerated D1 turnover but functional PSII

  • In Chlamydomonas, the psb28 mutant is more severely impaired in PSII assembly

  • These differences suggest adaptations of Psb28 function to the specific architecture and assembly pathways of photosystems in different organisms

These findings indicate that while the core function of Psb28 in PSII assembly is conserved, its precise role and importance may vary between different photosynthetic organisms, potentially reflecting adaptations to different ecological niches and photosynthetic strategies.