研究目的
To determine the binding sites for L-phenylalanine in TyrR protein via a rational mutation analysis combining biosensors and computer-aided simulation.
研究成果
Residues at positions 160, 173, and 184 of TyrR protein are the binding sites for L-Phe, with 173 and 160 providing hydrogen bonds and 184 making space for the benzene ring. Mutations at these sites reduce activation without affecting repression, indicating specific binding roles.
研究不足
Technical difficulties and high cost of analysis for protein-ligand interactions; mutations may affect protein conformation; biosensor sensitivity plateaued at high dipeptide concentrations.
1：Experimental Design and Method Selection:
Combined computer-aided prediction methods (SiteMap and Glide for docking, AMBER16 for molecular dynamics simulation) and biosensors (GFP-based reporter system) to identify ligand-binding sites.
2：Sample Selection and Data Sources:
Used E. coli strains (MG1655, TB1, TB2, B01 mutant), plasmids (pSenphe, pSenphe-12 derivatives), and genomic DNA.
3：List of Experimental Equipment and Materials:
Included multimode microplate reader for fluorescence measurement, HPLC for L-Phe concentration, PCR and Gibson assembly for plasmid construction, DpnI enzyme, and software like Schrodinger Maestro.
4：Experimental Procedures and Operational Workflow:
Constructed biosensor plasmids, performed site-directed mutagenesis, cultured cells in M9 minimal medium, induced with IPTG and Phe-Phe dipeptide, measured GFP fluorescence and β-galactosidase activity, conducted molecular docking and MD simulations.
5：Data Analysis Methods:
Analyzed fluorescence data with spectrometry, used Miller units for β-galactosidase assay, performed cluster analysis on MD trajectories.
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DpnI enzyme
Thermo-Fisher Scientific
Digestion of DNA for plasmid construction
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multimode microplate reader
Measurement of GFP fluorescence
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HPLC
Measurement of intracellular L-Phe concentration
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Schrodinger Maestro
Maestro 2018
Schrodinger
Protein structure preparation and molecular docking
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SiteMap
SiteMap 2018
Schrodinger
Mapping potential binding pockets in protein structures
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Glide
Glide 2018
Schrodinger
Molecular docking experiments
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AMBER16
Energy minimization and molecular dynamics simulation
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