Abstract
The putativedehelix of the D1 protein is located at the acceptor side of photosystem II (PS II) and serves as an indispensable part of a niche that binds the secondary plastoquinone QB. Combinatorial mutagenesis was applied to a stretch of four residues in a highy conserved region of this putative helix in order to reveal amino acid combinations that are able to support PS II function. An obligate photoheterotrophic mutant of the cyanobacteriumSynechocystiss o. PCC 6803, missing four residues (ΔYFGR254-7in thedehelix, was transformed with a D1-coding sequence carrying fully degenerate combinations of codons at the site of the deletion. Upon selection for photoautotrophy, 25 mutants with functional PS II were isolated. All mutants showed different codon combinations at positions 254 to 257; none was identical to the wild-type sequence, and none of the conserved residues was found to be mandatory for PS II function. However, 24 of the mutants contained Tyr or Phe at position 254 while at the other three positions many different amino acid combinations could be functionally accommodated. Most sequences maintained an amphiphilic arrangement of the helix that may align Tyr254 facing the lQBbinding pocket. This residue is proposed to be functionall y analogous to Phe216 of the L subunit in purple bacteria which contributes to binding of QB. Most of the PS II properties were similar in the mutants compared to wild-type. Noticeable modifications in the mutants concerned the semiquinone equilibrium of electron transfer between QAand QB, and the affinity of PS II inhibitors. Differential effects on the semiquinone equilibrium were observed between two distinct quinones occupying the QBsite (plastoquinoneversus2,5-dichloro-p-benzo-quinone), implying that residues in this domain are involved, directly or indirectly, with different binding determinants of the quinones. Even though many different combinations of amino acids in positions 254 to 257 of the D1 protein may satisfy the primary function of PS II, complex requirements need to be combined for optimized performance of the QBbinding niche.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 120-131 |
| Number of pages | 12 |
| Journal | Journal of Molecular Biology |
| Volume | 246 |
| Issue number | 1 |
| DOIs | |
| State | Published - Feb 10 1995 |
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Keywords
- combinatorial mutagenesis
- cyanobacteria
- molecular evolution
- protein-cofactor interaction
- reaction center
ASJC Scopus subject areas
- Molecular Biology
- Virology
Cite this
Many Combinations of Amino Acid Sequences in a Conserved Region of the D1 Protein Satisfy Photosystem II Function. / Kless, Hadar; Vermaas, Willem.
In: Journal of Molecular Biology, Vol. 246, No. 1, 10.02.1995, p. 120-131.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Many Combinations of Amino Acid Sequences in a Conserved Region of the D1 Protein Satisfy Photosystem II Function
AU - Kless, Hadar
AU - Vermaas, Willem
PY - 1995/2/10
Y1 - 1995/2/10
N2 - The putativedehelix of the D1 protein is located at the acceptor side of photosystem II (PS II) and serves as an indispensable part of a niche that binds the secondary plastoquinone QB. Combinatorial mutagenesis was applied to a stretch of four residues in a highy conserved region of this putative helix in order to reveal amino acid combinations that are able to support PS II function. An obligate photoheterotrophic mutant of the cyanobacteriumSynechocystiss o. PCC 6803, missing four residues (ΔYFGR254-7in thedehelix, was transformed with a D1-coding sequence carrying fully degenerate combinations of codons at the site of the deletion. Upon selection for photoautotrophy, 25 mutants with functional PS II were isolated. All mutants showed different codon combinations at positions 254 to 257; none was identical to the wild-type sequence, and none of the conserved residues was found to be mandatory for PS II function. However, 24 of the mutants contained Tyr or Phe at position 254 while at the other three positions many different amino acid combinations could be functionally accommodated. Most sequences maintained an amphiphilic arrangement of the helix that may align Tyr254 facing the lQBbinding pocket. This residue is proposed to be functionall y analogous to Phe216 of the L subunit in purple bacteria which contributes to binding of QB. Most of the PS II properties were similar in the mutants compared to wild-type. Noticeable modifications in the mutants concerned the semiquinone equilibrium of electron transfer between QAand QB, and the affinity of PS II inhibitors. Differential effects on the semiquinone equilibrium were observed between two distinct quinones occupying the QBsite (plastoquinoneversus2,5-dichloro-p-benzo-quinone), implying that residues in this domain are involved, directly or indirectly, with different binding determinants of the quinones. Even though many different combinations of amino acids in positions 254 to 257 of the D1 protein may satisfy the primary function of PS II, complex requirements need to be combined for optimized performance of the QBbinding niche.
AB - The putativedehelix of the D1 protein is located at the acceptor side of photosystem II (PS II) and serves as an indispensable part of a niche that binds the secondary plastoquinone QB. Combinatorial mutagenesis was applied to a stretch of four residues in a highy conserved region of this putative helix in order to reveal amino acid combinations that are able to support PS II function. An obligate photoheterotrophic mutant of the cyanobacteriumSynechocystiss o. PCC 6803, missing four residues (ΔYFGR254-7in thedehelix, was transformed with a D1-coding sequence carrying fully degenerate combinations of codons at the site of the deletion. Upon selection for photoautotrophy, 25 mutants with functional PS II were isolated. All mutants showed different codon combinations at positions 254 to 257; none was identical to the wild-type sequence, and none of the conserved residues was found to be mandatory for PS II function. However, 24 of the mutants contained Tyr or Phe at position 254 while at the other three positions many different amino acid combinations could be functionally accommodated. Most sequences maintained an amphiphilic arrangement of the helix that may align Tyr254 facing the lQBbinding pocket. This residue is proposed to be functionall y analogous to Phe216 of the L subunit in purple bacteria which contributes to binding of QB. Most of the PS II properties were similar in the mutants compared to wild-type. Noticeable modifications in the mutants concerned the semiquinone equilibrium of electron transfer between QAand QB, and the affinity of PS II inhibitors. Differential effects on the semiquinone equilibrium were observed between two distinct quinones occupying the QBsite (plastoquinoneversus2,5-dichloro-p-benzo-quinone), implying that residues in this domain are involved, directly or indirectly, with different binding determinants of the quinones. Even though many different combinations of amino acids in positions 254 to 257 of the D1 protein may satisfy the primary function of PS II, complex requirements need to be combined for optimized performance of the QBbinding niche.
KW - combinatorial mutagenesis
KW - cyanobacteria
KW - molecular evolution
KW - protein-cofactor interaction
KW - reaction center
UR - http://www.scopus.com/inward/record.url?scp=0028902008&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028902008&partnerID=8YFLogxK
U2 - 10.1006/jmbi.1994.0071
DO - 10.1006/jmbi.1994.0071
M3 - Article
C2 - 7853392
AN - SCOPUS:0028902008
VL - 246
SP - 120
EP - 131
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 1
ER -