Ultrasound-Synchronized In Situ Crystallization Locks Transient High-Activity Enzyme Conformations within Covalent Organic Frameworks

Abstract

Enzymes transiently access high-activity conformations with exposed cryptic sites and heightened substrate affinity, yet these states dissipate upon relaxation to the thermodynamic ground state once the activating perturbation is removed, leaving this catalytic potential largely inaccessible. Here we show that such fleeting conformations can be captured by synchronizing enzyme activation with host crystallization in a single sonochemical process, where ultrasound simultaneously drives enzymes into open conformations and promotes aqueous crystallization of covalent organic frameworks that grow around and lock the activated proteins before relaxation occurs. Using a library of six frameworks with systematically varied pore sizes and chemistries, we find that effective locking requires both tight steric confinement and abundant nonbonded interactions. The optimal framework increased the catalytic activity of horseradish peroxidase by 3.4-fold, primarily through a marked reduction in the Michaelis constant, while retaining >70% activity over 10 catalytic cycles. Molecular dynamics simulations identify the captured species as a globally reorganized open state defined by displacement of gating phenylalanine residues, while high-resolution 2D solid-state NMR analysis further elucidates the COF-enzyme interfacial interactions that stabilize this otherwise metastable conformation. Moreover, ex situ controls in which activation precedes encapsulation fail entirely, establishing that temporal synchrony between conformational opening and framework crystallization is mechanistically essential. Extending this strategy to laccase, cytochrome c, and lipase enhanced immobilized enzyme activity by 3.8-fold, 2.7-fold, and 1.5-fold over conventional stirring. This work reframes enzyme immobilization from passive protection to active conformational upgrading, enabling a general strategy to harvest transient functional states beyond equilibrium reach.