Proteases are involved in essential biological functions in nature and have become drug targets recently. In spite of the promising progress, two challenges, (i) the intrinsic instability and (ii) the difficulty in monitoring the catalytic process in real time, still hinder the further understanding and engineering of protease functionalities. These challenges are caused by the lack of proper materials/approaches to stabilize proteases and monitor proteolytic products (truncated polypeptides) in real time in a highly heterogeneous reaction mixture. This work combines metal−organic materials (MOMs), site-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy, and mass spectrometry (MS) to overcome both barriers. A model protease, trypsin, which cleaves the peptide bonds at lysine or arginine residues, was immobilized on a Ca-MOM via aqueous-phase, one-pot cocrystallization, which allows for trypsin protection and ease of separation from its proteolytic products. Time-resolved EPR and MS were employed to monitor the populations, rotational motion, and sequences of the cleaved peptide truncations of a model protein substrate as the reaction proceeded. Our data suggest a significant (at least 5−10 times) enhancement in the catalytic efficiency (k cat /k m ) of trypsin@Ca-MOM and excellent reusability as compared to free trypsin in solution. Surprisingly, entrapping trypsin in Ca-MOMs results in cleavage site/region selectivity against the protein substrate, as compared to the near nonselective cleavage of all lysine and arginine residues of the substrate in solution. Remarkably, immobilizing trypsin allows for the separation and, thus, MS study on the sequences of truncated peptides in real time, leading to a time-resolved "movie" of trypsin proteolysis. This work demonstrates the use of MOMs and cocrystallization to enhance the selectivity, catalytic efficiency, and stability of trypsin, suggesting the possibility of tuning the catalytic performance of a general protease using MOMs.

Aqueous-phase co-crystallization (also known as biomimetic mineralization or biomineralization) is a unique way to encapsulate large enzymes, enzyme clusters, and enzymes with large substrates in metal–organic frameworks (MOFs), broadening the application of MOFs as enzyme carriers. The crystallinity of resultant enzyme@MOF biocomposites, however, can be low, raising a concern about how MOF crystal packing quality affects enzyme performance upon encapsulation. The challenges to overcome this concern are (1) the limited database of enzyme performance upon biomineralization in different aqueous MOFs and (2) the difficulty in probing enzyme restriction and motion in the resultant MOF scaffolds, which are related to the local crystal packing quality/density, under the interference of the MOF backgrounds. We have discovered several new aqueous MOFs for enzyme biomineralization with varied crystallinity [Expanding the Library of Metal–Organic Frameworks (MOFs) for Enzyme BiomineralizationJordahlD.ArmstrongZ.LiQ.GaoR.LiuW.JohnsonK.BrownW.ScheiwillerA.FengL.UgrinovA.MaoH.ChenB.QuadirM.PanY.LiH.YangZ. Jordahl, D. Armstrong, Z. Li, Q. Gao, R. Liu, W. Johnson, K. Brown, W. Scheiwiller, A. Feng, L. Ugrinov, A. Mao, H. Chen, B. Quadir, M. Pan, Y. Li, H. Yang, Z. ACS Appl. Mater. Interfaces2022145161951629]. Here, we address the second challenge by probing enzyme dynamics/restriction in these MOFs at the residue level via site-directed spin labeling (SDSL)–electron paramagnetic resonance (EPR) spectroscopy, a unique approach to determine protein backbone motions regardless of the background complexity. We encapsulated a model large-substrate enzyme, lysozyme, in eight newly discovered MOFs, which possess various degrees of crystallization, via aqueous-phase co-crystallization. Through the EPR study and simulations, we found rough connections between (a) enzyme mobility/dynamics and MOF crystal properties (packing quality and density) and (b) enzyme areas exposed above each MOF and their catalytic performance. This work suggests that protein SDSL and EPR can serve as an indicator of MOF crystal packing quality/density when biomineralized in MOFs. The method can be generalized to probing the dynamics of other enzymes on other solid surfaces/interfaces and guide the rational design of solid platforms (ca. MOF...

Confining enzymes in well-shaped MOF compartments is a promising approach to mimic the cellular environment of enzymes and determine enzyme structure-function relationship therein. Under the cellular crowding, however, enzymes can...