研究目的
To investigate site-resolved vibrational energy transfer (VET) in proteins using a genetically encoded ultrafast heater, specifically the non-canonical amino acid β-(1-azulenyl)-L-alanine (AzAla), to provide experimental evidence for VET pathways linked to allosteric communication.
研究成果
The research successfully demonstrated site-resolved VET in a protein using genetically encoded AzAla, with energy transfer occurring on picosecond timescales. This approach enables detailed mapping of VET pathways, potentially elucidating their role in allosteric communication and protein function, and opens avenues for broader applications in protein studies.
研究不足
The study is limited to specific protein systems (PDZ3 domain) and positions; generalizability to other proteins requires further validation. The use of E. coli expression may not be suitable for all proteins, and the technique relies on the availability of orthogonal aaRS-tRNA pairs. Potential perturbations from ncAA incorporation and the need for high-yield protein production are constraints.
1：Experimental Design and Method Selection:
The study involved genetic code expansion to site-specifically incorporate AzAla into proteins, using an engineered aminoacyl-tRNA synthetase (aaRS) for AzAla. Ultrafast IR spectroscopy was employed to monitor VET from AzAla to a sensor amino acid (L-azidohomoalanine, Aha) in a protein-peptide complex.
2：Sample Selection and Data Sources:
The PDZ3 domain of PSD-95 protein was used as a model system, with AzAla incorporated at positions Phe325 and Phe
3：A peptide ligand (YKQTAhaV) containing Aha at position P-1 was synthesized and bound to PDZ. Proteins were expressed in E. coli BL21(DE3) cells. List of Experimental Equipment and Materials:
3 Equipment included SDS-PAGE for protein analysis, ESI-MS for mass confirmation, isothermal titration calorimetry (ITC) for binding assays, and an ultrafast IR spectroscopy setup with 613 nm pump pulses and IR probe pulses around 2120 cm?1. Materials included AzAla, Aha, and various biochemical reagents.
4：Experimental Procedures and Operational Workflow:
AzAlaRS was engineered from MjTyrRS through library construction and selection. Proteins were expressed, purified via immobilized metal-ion affinity chromatography (IMAC), and characterized. VET measurements involved exciting AzAla with 613 nm light and probing the azide stretch of Aha with IR pulses, recording time-resolved signals.
5：Data Analysis Methods:
Data were analyzed to extract VET timescales and signal amplitudes, with comparisons to molecular dynamics simulations and control experiments without the sensor.
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