研究目的
Investigating the structure and binding efficiency relations of QB site inhibitors in photosynthetic reaction centres to understand how herbicide molecular structure, interactions with the quinone binding site, and protein environment affect inhibition efficiency.
研究成果
The herbicide inhibition efficiency is governed by steric and electronic factors. Apolar bulky groups on the N-7 atom enhance binding, while similar groups on N-8 are detrimental. Nitrogen atoms with high electron density facilitate stronger hydrogen bonds with protein amino acids. These insights can aid in designing more effective herbicides and biosensors for environmental monitoring.
研究不足
The study is limited to bacterial reaction centres (e.g., from Rhodobacter sphaeroides and Blastochloris viridis), and may not fully extrapolate to other photosynthetic systems. Variability in detergent concentrations and lipid environments could affect binding constants. Computational models rely on approximations and may not capture all molecular interactions accurately.
1：Experimental Design and Method Selection:
The study involved determining inhibition constants (KI) and binding enthalpy values (?Hbind) for various herbicides using kinetic spectrophotometry and molecular modeling. Herbicide titrations were performed on RC samples in detergent micelles and proteoliposomes to monitor inhibition of electron transfer. Molecular structures were optimized using semi-empirical and ab initio calculations.
2：Sample Selection and Data Sources:
Carotenoid-less Rb. sphaeroides R-26 cells were grown and RCs were prepared. Herbicides (terbutryn, terbumeton, ametryn, prometon, prometryn, atrazine) were used as inhibitors. Crystal structures (2PRC, 1DXR, 5PRC) were downloaded from the Brookhaven Protein Data Bank.
3：List of Experimental Equipment and Materials:
Equipment included a single-beam kinetic spectrophotometer (local design), centrifuge filter (Whatman VectaSpin 3, 10 kDa exclusion), and software like GAMESS US, HyperChem 7.0, Maestro 9.3.5. Materials included DDAO detergent (Fluka), DEAE-Sephacel (Sigma), UQ-10 (Sigma), Triton X-100 (Sigma), phospholipids (PC, PG, CL from Sigma), and herbicides (Fluka).
4：0, Maestro Materials included DDAO detergent (Fluka), DEAE-Sephacel (Sigma), UQ-10 (Sigma), Triton X-100 (Sigma), phospholipids (PC, PG, CL from Sigma), and herbicides (Fluka). Experimental Procedures and Operational Workflow:
4. Experimental Procedures and Operational Workflow: RCs were solubilized and purified. QB site was reconstituted with UQ-10. Herbicide titrations were done by adding small volumes of herbicide stock solutions to RC samples. Flash-induced absorption changes at 860 nm were measured to monitor charge recombination. Proteoliposomes were prepared by incorporating RCs into lipid vesicles. Molecular modeling involved geometry optimization and charge distribution calculations.
5：Herbicide titrations were done by adding small volumes of herbicide stock solutions to RC samples. Flash-induced absorption changes at 860 nm were measured to monitor charge recombination. Proteoliposomes were prepared by incorporating RCs into lipid vesicles. Molecular modeling involved geometry optimization and charge distribution calculations. Data Analysis Methods:
5. Data Analysis Methods: Data were fitted using Michaelis-Menten kinetic models to determine KI values. Mulliken population analysis was used for charge distributions. Statistical analysis included error estimations from fits.
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kinetic spectrophotometer
local design
Measuring flash-induced absorption changes at 860 nm to monitor charge recombination in RC samples.
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centrifuge filter
VectaSpin 3
Whatman
Concentrating RC samples by filtration.
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DDAO detergent
Fluka
Solubilizing and purifying RCs.
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DEAE-Sephacel
Sigma
Anion-exchange chromatography for RC purification.
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UQ-10
Sigma
Reconstituting the QB site in RCs.
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Triton X-100
Sigma
Solubilizing UQ-10 in aqueous solutions.
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phospholipids
PC, PG, CL
Sigma
Preparing proteoliposomes for RC incorporation.
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herbicides
terbutryn, terbumeton, ametryn, prometon, prometryn, atrazine
Fluka
Inhibiting electron transfer in RCs.
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GAMESS US software
Performing quantum chemical calculations for molecular charge distributions.
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HyperChem
7.0
Constructing graphs and calculating binding distances for molecular modeling.
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Maestro
9.3.5
Schr?dinger, LLC
Preparing protein structures and creating ligand interaction diagrams.
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