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 [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. Expanding the Library of Metal-Organic Frameworks (MOFs) for Enzyme Biomineralization. ACS Appl. Mater. Interfaces 2022, 14 (46), 51619-51629, DOI: 10.1021/acsami.2c12998]. 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. MOFs) to customize enzyme immobilization.
Keywords: electron paramagnetic resonance (EPR); enzyme encapsulation; enzyme orientation; metal−organic frameworks (MOFs); site-directed spin labeling (SDSL).