Combined Mutagenesis and Kinetics Characterization of the Bilin-Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803
| Autor: | Dan Miao, Lorena Valle, Alexander Gutt, Claudio D. Borsarelli, Sarah Raffelberg, Kun Tang, Wolfgang Gärtner, Kai-Hong Zhao, Jonas Mechelke, Xiu Ling Xu |
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| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
Models
Molecular Histidine Kinase Otras Ciencias Biológicas Protein Data Bank (RCSB PDB) Molecular Conformation PHYCOCYANOBILIN Biochemistry Absorbance Ciencias Biológicas chemistry.chemical_compound GLOBAL FIT Phycocyanobilin Bacterial Proteins PAS domain Phycobilins TIME RESOLVED Molecular Biology MUTAGENESIS Phytochrome biology Organic Chemistry Synechocystis Phycocyanin Chromophore biology.organism_classification Protein Structure Tertiary PHYTOCHROMES Crystallography Kinetics chemistry Mutagenesis Site-Directed Molecular Medicine Cyanobacteriochrome CHROMOPHORES Protein Kinases CIENCIAS NATURALES Y EXACTAS |
| Popis: | The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (lmax=649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 ms, 390 ms, and 1.5 ms) for the red-to-green conversion, and 1.2 ms, 340 ms, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp.PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the greenabsorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation. Fil: Xu, Xiu Ling. Max-Planck-Institute for Chemical Energy Conversion; Alemania Fil: Gutt, Alexander. Max-Planck-Institute for Chemical Energy Conversion; Alemania Fil: Mechelke, Jonas. Max-Planck-Institute for Chemical Energy Conversion; Alemania Fil: Raffelberg, Sarah. Max-Planck-Institute for Chemical Energy Conversion; Alemania Fil: Tang, Kun. Max-Planck-Institute for Chemical Energy Conversion; Alemania. Huazhong Agricultural University; República de China Fil: Miao, Dan. Huazhong Agricultural University; República de China Fil: Valle, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina Fil: Borsarelli, Claudio Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina Fil: Zhao, Kai Hong. Huazhong Agricultural University; República de China Fil: Gärtner, Wolfgang. Max-Planck-Institute for Chemical Energy Conversion; Alemania |
| Databáze: | OpenAIRE |
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