Site-directed mutagenesis of the CP 47 protein of photosystem II: 167W in the lumenally exposed loop C is required for photosystem II assembly and stability

Wu, Jituo; Putnam-Evans, Cindy; Bricker, Terry M.

doi:10.1007/bf00019106

Cited by 15 publications

(3 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A number of studies in Synechocystis have focused on the importance of loop domains for protein function of the chlbinding proteins CP47, D1, and D2 of photosystem II. These studies include deletions and point mutations in loop c (25) and loop e (26-28) of CP47, a lumenal domain of D2 (22), and a stromal (29) and a lumenal (30) domain of D1. Several of these mutations rendered the organisms nonphotosynthetic.…”

Section: Discussionmentioning

confidence: 99%

Random Mutations Directed to Transmembrane and Loop Domains of the Light-Harvesting Chlorophyll a/b Protein: Impact on Pigment Binding

Heinemann

Paulsen

1999

Biochemistry

View full text Add to dashboard Cite

The major light-harvesting complex of photosystem II (LHCII) can be reconstituted in vitro by folding its bacterially expressed apoprotein, Lhcb, in detergent solution in the presence of chlorophylls and carotenoids. To compare the impact of alpha-helical transmembrane domains and hydrophilic loop domains of the apoprotein on complex formation and stability, we introduced random mutations into a segment of the protein comprising the stromal loop, the third (C-proximal) transmembrane helix, and part of the amphipathic helix in the C-terminal domain. The mutant versions of Lhcb were screened for the loss of their ability to form stable LHCII upon reconstitution in vitro. Most steps during the screening, including expression of the recombinant protein, its reconstitution with pigments, and the assay for complex formation by measuring energy transfer from chlorophyll b to chlorophyll a, were performed as one-vessel reactions on 96-well microtiter plates. This enabled us to screen several hundred mutant Lhcb versions. Mutants that had lost their ability to form stable LHCII carried between one and four amino acid exchanges. Among the single-point mutations, several were at positions in the C-proximal transmembrane helix, including an amino acid that is thought to be directly involved in chlorophyll binding. However, we also found four point mutations in the stromal loop domain that, in our assay, completely abolished the formation of stable LHCII. These data show that the stromal loop domain has a significant impact on LHCII formation and/or stability in vitro.

show abstract

Section: Discussionmentioning

confidence: 99%

Random Mutations Directed to Transmembrane and Loop Domains of the Light-Harvesting Chlorophyll a/b Protein: Impact on Pigment Binding

Heinemann

Paulsen

1999

Biochemistry

View full text Add to dashboard Cite

show abstract

“…A similar mechanism may occur in the R305S and R342S mutants described here. The apparent protective effect on oxygen-evolving activity seen with the R342S mutant grown in the presence of DCMU may result from a reduction of damaging oxidizing-side radicals in the presence of this PSII inhibitor ( , ).…”

Section: Discussionmentioning

confidence: 99%

Site-Directed Mutagenesis of Basic Arginine Residues 305 and 342 in the CP 43 Protein of Photosystem II Affects Oxygen-Evolving Activity in Synechocystis 6803

1998

Self Cite

View full text Add to dashboard Cite

The intrinsic chlorophyll protein CP 43, a component of photosystem II (PS II) in higher plants, green algae, and cyanobacteria, is encoded by the psbC gene. Oligonucleotide-directed mutagenesis was employed to introduce mutations into a segment of psbC that encodes the large extrinsic loop E of CP 43 in the cyanobacterium Synechocystis 6803. Two mutations, R305S and R342S, each produced a strain with impaired photosystem II activity. The R305S mutant strain grew photoautotrophically at rates comparable to the control strain. Immunological analyses of a number of PSII components indicated that this mutant accumulated normal quantities of PSII proteins. However, this mutant evolved oxygen to only 70% of control rates at saturating light intensities. Measurements of total variable fluorescence yield indicated that this mutant assembled approximately 70% of the PSII centers found in the control strain. The R342S mutant failed to grow photoautotrophically and exhibited no capacity for oxygen evolution. However, when grown photoheterotrophically in medium containing both glucose and 3-(3, 4-dichlorophenyl)-1,1-dimethylurea (DCMU), oxygen-evolving activity was observed in the R342S mutant, but at a low level of approximately 10% of the control rate. Immunological analysis of isolated thylakoid membranes from this mutant also indicated that this strain accumulated normal amounts of PSII core proteins. Total variable fluorescence yields for the R342S mutant indicated that it assembled a severely reduced number of fully functional PSII centers. R305S and R342S mutant strains exhibited, respectively, 2.7- and 4-fold increased sensitivity to photoinactivation. The fluorescence rise times for both mutants were comparable to the control when hydroxylamine was used as electron donor. However, both strains exhibited an increase (2.5- and 8-fold, respectively, for R305S and R342S) in fluorescence rise times with water as an electron donor. These results suggest that the mutations R305S and R342S each produce a defect associated with the oxygen-evolving complex of photosystem II. These are the first site-directed mutations in CP 43 to show such an effect.

show abstract

“…The second mutant, R448G, has an increased chloride requirement and is an obligate photoheterotroph under chloride-limiting conditions (24). In loop C, alteration of a single amino acid residue (W167S) resulted in 75% loss in oxygen evolution capacity and the mutant accumulated very few PS II reaction centers (27).…”

mentioning

confidence: 99%

Functional Analysis of Combinatorial Mutants Altered in a Conserved Region in Loop E of the CP47 Protein in Synechocystis sp. PCC 6803

Tichý

Vermaas

1998

Biochemistry

View full text Add to dashboard Cite

Regions in the large lumenally exposed region (loop E) of CP47 affect properties of the watersplitting system in photosystem II (PS II). To investigate the role of these regions, we developed a method for functional complementation of obligate photoheterotrophic mutants carrying a deletion in one such region. Using an obligate photoheterotrophic mutant that carries a short deletion (delta (D440-P447) in loop E of CP47, completely degenerate sequences of eight codons in length were introduced at the site of the deletion. Transformants that were complemented to photoautotrophic growth were selected, and 20 such mutants were studied. Sequence analysis revealed that, as expected, in each of them CP47 had been restored to its wild-type length. However, none of the amino acid residues in the deleted region were found to be critical for function. A negatively charged residue at position 440 and a positively charged one at position 444 were favored but not required. Photoautotrophic growth of mutants obtained varied from almost normal to significantly impaired. The mutants contained 20-100% of the amount of PS II present in the wild type, with PS II amounts correlating with the initial rates of oxygen evolution. The mutants had a high rate of photoinactivation, and many mutants showed an up to 1000-fold increase in chloride requirement for photoautotrophic growth. These phenotypic effects were a direct consequence of the CP47 mutations and were not caused by altered binding of one of the extrinsic proteins. No particular amino acid residues in positions 440-447 of CP47 were found to be indispensable for photoautotrophic growth, and many amino acid combinations in this region support PS II function. However, the mutagenized region is shown to interact with the oxygen-evolving site of PS II and appears to have a direct role in chloride binding.

show abstract

Site-directed mutagenesis of the CP 47 protein of photosystem II: 167W in the lumenally exposed loop C is required for photosystem II assembly and stability

Cited by 15 publications

References 30 publications

Random Mutations Directed to Transmembrane and Loop Domains of the Light-Harvesting Chlorophyll a/b Protein: Impact on Pigment Binding

Random Mutations Directed to Transmembrane and Loop Domains of the Light-Harvesting Chlorophyll a/b Protein: Impact on Pigment Binding

Site-Directed Mutagenesis of Basic Arginine Residues 305 and 342 in the CP 43 Protein of Photosystem II Affects Oxygen-Evolving Activity in Synechocystis 6803

Functional Analysis of Combinatorial Mutants Altered in a Conserved Region in Loop E of the CP47 Protein in Synechocystis sp. PCC 6803

Contact Info

Product

Resources

About