A Dual-Key Lock Strategy for Bright Fluorescent Proteins Fabrication via Synergistic Noncovalent Activation of Aggregation-Induced Emission Luminogen

Abstract The development of simple, bio-friendly strategies to engineer bright fluorescent proteins (FPs) is crucial for biosensing and bioimaging. Conventional synthesis of FPs requires time-consuming chromophore maturation and tedious preparation, while covalent conjugation methods often involve long reactions and risk loss of bioactivity. Herein, we propose a noncovalent “Dual-Key Lock” strategy and demonstrated the strategy using a selective aggregation-induced emission luminogen (AIEgen), TCBPE. This mechanism relies on the synergistic action of hydrogen bonding and hydrophobic interactions, which collectively confine TCBPE within the binding pocket of bovine serum albumin (BSA@TCBPE), effectively restricting intramolecular motion to activate AIE. Controlled studies with two reference AIEgens (TPE, TCPE) and molecular docking simulations validated this synergistic action with TCBPE. Based on strong noncovalent binding affinity (Kd = 54.8 nM), the fluorescence intensity of BSA@TCBPE reached 92% of its maximum value at 5 min and exhibited 12.62-fold enhancement over free TCBPE. BSA@TCBPE had a high quantum yield of 73.74% and excellent photostability under irradiation at 2 W/cm2 for 90 min. Importantly, the noncovalent conjugation preserved the native functionality of BSA, enabling its effective use in biosensing, while also facilitating high-contrast cellular imaging with low cytotoxicity. This work elucidated key supramolecular interactions and established a simple and efficient platform for constructing high-performance fluorescent proteins with biofunctional applications.