Probing structure–function relationships in early events in photosynthesis using a chimeric photocomplex
| Autor: | Kenji V. P. Nagashima, Mai Sasaki, Kazuhito Inoue, Takashi Ohno, Zheng-Yu Wang-Otomo, Kanako Hashimoto, Akira Yamaguchi, Mizuki Takenouchi, Jian Ren Shen, Kenta Gotou, Yukihiro Kimura, Long Jiang Yu, Shinichi Takaichi, Michael T. Madigan, Yuuta Shibuya, Tomoaki Kawakami, Yuto Abe, Sakiko Nagashima |
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| Rok vydání: | 2017 |
| Předmět: |
Models
Molecular 0301 basic medicine Photosynthetic reaction centre Protein Conformation Stereochemistry Photosynthetic Reaction Center Complex Proteins Mutant Resonance Raman spectroscopy Light-Harvesting Protein Complexes Rhodobacter sphaeroides Chromatiaceae Structure-Activity Relationship 03 medical and health sciences Bacterial Proteins Gene cluster Photosynthesis Binding Sites Multidisciplinary 030102 biochemistry & molecular biology biology Thermophile Mutagenesis Biological Sciences biology.organism_classification Carotenoids Fluorescence 030104 developmental biology Biochemistry Calcium Protein Binding |
| Zdroj: | Proceedings of the National Academy of Sciences. 114:10906-10911 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium Thermochromatium tepidum requires Ca2+ for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the Tch. tepidum LH1 complex in an engineered Rhodobacter sphaeroides mutant strain. This system contained a chimeric pufBALM gene cluster (pufBA from Tch. tepidum and pufLM from Rba. sphaeroides) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed Tch. tepidum wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in Rba. sphaeroides) was incorporated into the Tch. tepidum LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αβ-subunit. The hybrid LH1-RC complexes expressed in Rba. sphaeroides were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, β-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the Tch. tepidum LH1 complex; in contrast, α-N50 does not participate in Ca2+ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated Tch. tepidum LH1-RC complex and have unambiguously identified the location of Ca2+ within this key antenna complex. |
| Databáze: | OpenAIRE |
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