Recombinant Sorghum bicolor Photosystem II reaction center protein H (psbH) is a genetically engineered version of the native Photosystem II (PSII) subunit H, produced in heterologous systems like E. coli. This 10 kDa phosphoprotein is critical for PSII assembly, stability, and function in plants. Its recombinant form enables structural, functional, and biochemical studies of PSII, a key component of light-dependent photosynthesis in chloroplasts.
Gene Name: psbH
Protein Name: PSII-H, Photosystem II 10 kDa phosphoprotein
Recombinant psbH is synthesized in E. coli using genetic engineering, with the native psbH gene cloned into expression vectors. The His-tag enables affinity purification via nickel columns.
Studies in Chlamydomonas reinhardtii revealed that psbH is essential for PSII core dimerization. Deletion mutants showed impaired accumulation of PSII proteins, indicating its role in stabilizing the complex . Electron microscopy of His-tagged PSII core dimers localized psbH near cytochrome b(559), suggesting proximity to PSII’s electron transfer chain .
Phosphorylation of psbH may modulate PSII structure or activity. While specific sites remain unconfirmed, its phosphorylation is hypothesized to influence light-dependent reactions or stress responses .
Structural Studies: Recombinant psbH aids in mapping PSII subunit interactions via cross-linking or crystallography .
Functional Assays: Used to study PSII assembly defects or inhibitor mechanisms (e.g., sorgoleone derivatives) .
Organism | psbH UniProt ID | Sequence Length | Expression Host |
---|---|---|---|
Sorghum bicolor | A1E9V3 | 2–73 aa | E. coli |
Chaetosphaeridium globosum | Q8M9Z3 | 2–74 aa | E. coli |
Cyanidioschyzon merolae | Q85FZ2 | 1–64 aa | E. coli |
KEGG: sbi:4549220
STRING: 4558.Sb03g017590.1
The psbH protein is a small but essential subunit of the Photosystem II (PSII) complex, participating in the early stages of PSII biogenesis. It forms part of the reaction center (RC) which consists of D1, D2, PsbI, and cytochrome b559 subunits . Research in model plant systems indicates that psbH contributes to the assembly, stability, and repair of the PSII reaction center.
Methodologically, psbH function can be studied through:
Mutation analysis using CRISPR-Cas9 or traditional mutagenesis techniques
Protein-protein interaction studies (co-immunoprecipitation, Y2H, BiFC)
Functional complementation experiments in psbH-deficient mutants
Chlorophyll fluorescence measurements to assess PSII activity
In plant chloroplasts, psbH is typically part of a polycistronic transcript derived from the psbB-psbT-psbH-petB-petD gene cluster . The processing of this RNA is influenced by RNA-binding proteins like HCF107, which impacts the metabolism of the polycistronic RNA and affects psbH expression .
For studying psbH regulation in Sorghum bicolor, researchers should employ:
RNA extraction and Northern blotting to detect transcript levels
5' RACE to map transcript termini
RNA-seq to quantify expression across tissues or conditions
RNA stability assays to determine transcript half-life
HCF107 has been shown to bind to the 5' UTR of psbH mRNA, protecting it from 5'→3' RNA degradation while also enhancing translation efficiency .
The ultra-high-density consensus genetic map for Sorghum bicolor provides valuable resources for genetic studies of psbH. This map integrates data from multiple mapping populations, resulting in 3,449 non-redundant polymorphic markers across the ten sorghum chromosomes, spanning 1,571.68 cM with an average of one marker per 0.46 cM .
For methodological approaches, researchers should:
Use the consensus genetic map to locate psbH and identify nearby markers
Reference the Sorghum bicolor v3.1.1 genome in Phytozome for sequence information
Leverage RNA-seq data for expression analysis in different tissues
Employ molecular markers for mapping studies or association genetics
HCF107 is a HAT (Half-A-TPR) protein that functions as a sequence-specific RNA binding protein affecting chloroplast gene expression . Research has revealed dual roles for HCF107:
RNA stabilization: HCF107 binds to the 5' end of processed psbH mRNA, protecting it from exonucleolytic degradation. Mutations in HCF107 cause the specific loss of processed RNAs with a 5' end 44 nt upstream of the psbH start codon .
Translation enhancement: HCF107 remodels RNA structure by binding to a region that would otherwise form an inhibitory duplex with the translation initiation region. This sequestration makes the region around the start codon more accessible to ribosomes .
Methodologically, this can be studied through:
RNA electrophoretic mobility shift assays to demonstrate protein-RNA binding
RNA structure probing methods like SHAPE analysis
In vitro translation assays with and without HCF107
Mutational analysis of binding sites in both the protein and RNA
ONE-HELIX PROTEIN1 (OHP1) and OHP2 play crucial roles in PSII assembly. Studies in Arabidopsis have shown that:
OHP1 and OHP2 are essential for the formation of the PSII reaction center
In their absence, synthesis of PSII core proteins D1/D2 and formation of the PSII RC is specifically blocked
These proteins form a complex with HIGH CHLOROPHYLL FLUORESCENCE244 (HCF244) along with D1, D2, PsbI, and cytochrome b559
This complex, termed the "PSII RC-like complex," appears to function at an early stage of PSII de novo assembly and during PSII repair under high-light conditions .
For researchers working with recombinant psbH, consideration should be given to:
Co-expression of OHP1 and OHP2 may improve incorporation of psbH into functional complexes
Mutagenesis of chlorophyll-binding residues in OHPs affects their function/stability, suggesting they may facilitate chlorophyll binding in vivo
Reconstitution experiments should consider the transient nature of the OHP-containing complexes
Post-translational modifications (PTMs) can significantly affect psbH function. To study these modifications in Sorghum bicolor:
Identification strategies:
Mass spectrometry-based proteomics (LC-MS/MS)
Phospho-specific antibodies for detecting phosphorylation
Chemical labeling techniques for specific modifications
Functional characterization:
Site-directed mutagenesis of modified residues
Biochemical assays comparing wild-type and mutant proteins
In vivo complementation studies using modified variants
Dynamics assessment:
Pulse-chase experiments to track modification timing
Light/dark transition studies to correlate with photosynthetic activity
Stress response experiments to identify regulation mechanisms
The choice of expression system significantly impacts the yield and functionality of recombinant psbH:
Expression System | Advantages | Disadvantages | Best Applications |
---|---|---|---|
E. coli (C41/C43) | High yield, ease of use, cost-effective | Limited post-translational modifications, potential inclusion body formation | Initial structural studies, antibody production |
Yeast (P. pastoris) | Eukaryotic PTMs, secretion capability, high density culture | Longer expression time, complex medium requirements | Functional studies requiring PTMs |
Insect cells | Complex eukaryotic PTMs, membrane protein expression | Higher cost, specialized equipment needed | Structural studies requiring native-like folding |
Chloroplast-based | Native environment, correct folding | Lower yield, technical complexity | Functional studies in quasi-native context |
Cell-free systems | Rapid, adaptable to toxic proteins | Higher cost, limited scale | Initial screening, directed evolution |
For membrane proteins like psbH, methodological considerations include:
Addition of detergents during extraction and purification
Optimization of induction conditions (temperature, inducer concentration)
Co-expression with chaperones to aid proper folding
Use of fusion partners to enhance solubility
Ensuring proper folding and functionality requires multiple complementary approaches:
Structural assessment:
Circular dichroism spectroscopy to analyze secondary structure
Limited proteolysis to assess conformational stability
Size-exclusion chromatography to evaluate oligomeric state
Functional assays:
Reconstitution with other PSII components
Oxygen evolution measurements if incorporated into larger PSII complexes
Binding studies with known interaction partners
Complex formation analysis:
Blue Native-PAGE to assess incorporation into complexes
Co-purification experiments with interaction partners
Electron microscopy of reconstituted complexes
In vivo validation:
Complementation of psbH-deficient mutants
Chlorophyll fluorescence measurements to assess PSII function
Growth assays under varying light conditions
Differentiating direct from indirect effects of psbH mutations requires systematic approaches:
Comparative mutation analysis:
Create a panel of mutations with varying biochemical properties
Compare phenotypes across mutation types
Identify patterns that correlate with specific functional domains
Time-course studies:
Monitor changes following inducible expression of mutant proteins
Early effects are more likely to be direct consequences
Late effects may represent compensatory or indirect responses
Multi-omics integration:
Combine transcriptomic, proteomic, and metabolomic analyses
Use network analysis to identify immediate vs. downstream effects
Apply statistical methods like partial correlation to separate direct and indirect links
In vitro reconstitution:
Purify components and reconstitute systems with wild-type or mutant psbH
Effects observed in reconstituted systems are more likely direct
Compare with in vivo observations to identify potential indirect effects
When facing contradictory results in psbH studies, researchers should:
Assess methodological differences:
Compare experimental conditions (temperature, light, growth media)
Evaluate differences in genetic backgrounds used
Consider expression system variations for recombinant studies
Perform targeted validation experiments:
Replicate key experiments using standardized protocols
Test critical hypotheses with alternative methodologies
Conduct side-by-side comparisons in a single laboratory
Consider biological context:
Evaluate developmental stages of plant material
Assess environmental conditions during experiments
Examine tissue-specific differences in psbH function
Statistical re-evaluation:
Perform meta-analysis of available data when possible
Increase biological replicates to improve statistical power
Apply more robust statistical methods appropriate for the data type
Purifying membrane proteins like psbH requires specialized approaches:
Detergent screening and selection:
Test multiple detergents (DDM, LMNG, digitonin) for optimal extraction
Assess protein stability in each detergent by size-exclusion chromatography
Consider detergent exchange during purification for downstream applications
Optimized purification workflow:
Membrane isolation by ultracentrifugation
Solubilization with selected detergent
Affinity chromatography using tagged protein
Size-exclusion chromatography for final polishing
Alternative approaches:
Styrene-maleic acid lipid particles (SMALPs) for detergent-free purification
Nanodiscs for reconstitution in a lipid environment
Amphipol stabilization for structural studies
Quality control methods:
SDS-PAGE and western blotting to verify purity
Mass spectrometry for precise molecular weight determination
Functional assays to confirm activity retention
To successfully reconstitute psbH into functional PSII complexes:
Component preparation:
Purify individual components under conditions that maintain native structure
Verify purity and functionality of each component before assembly
Consider co-expression of multiple components when appropriate
Assembly conditions:
Optimize buffer composition (pH, ionic strength, specific ions)
Select appropriate detergent or membrane mimetic environment
Control temperature and light conditions during assembly
Facilitated assembly approaches:
Validation methods:
Spectroscopic analysis to assess chlorophyll binding
Oxygen evolution measurements to confirm electron transport activity
Structural analysis by cryo-EM or other methods to verify complex formation