Random mutations directed to transmembrane and loop domains of the light-harvesting chlorophyll a/b protein: impact on pigment binding.
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.
Heinemann
B
B
Institut für Allgemeine Botanik, Universität Mainz, D-55099 Mainz, Germany.
Paulsen
H
H
D000595:Amino Acid Sequence; D002338:Carotenoids / Q000378:metabolism*; D002734:Chlorophyll / Q000378:metabolism*; D004735:Energy Transfer; D045342:Light-Harvesting Protein Complexes; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018532:Pisum sativum; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics* / Q000378:metabolism; D045332:Photosystem II Protein Complex; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013050:Spectrometry, Fluorescence
D045342:Light-Harvesting Protein Complexes; D008565:Membrane Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D002734:Chlorophyll; D002338:Carotenoids