Site-directed spin labeling-electron paramagnetic resonance spectroscopy in biocatalysis: Enzyme orientation and dynamics in nanoscale confinement

Expanding the “Library” of Metal–Organic Frameworks for Enzyme Biomineralization

“…13,19−30 Recently, researchers including our team have shown the possibility of using co-precipitation to remove the size limitation of the substrate, so that the entrapped enzymes are partially exposed to the reaction medium and partially buried under the MOF crystal surfaces, 31−35 as demonstrated on zeolitic imidazolate frameworks (ZIFs) and recent Ca-based metal−organic materials (MOMs, an analogue of MOFs but with a one-or two-dimensional structure); 31,33−35 the exposed enzyme regions were also revealed using our developed biophysical tools. 36,37 Importantly, our enzyme@Ca-MOM composites can be formed in the enzyme-friendly, aqueous phase under ambient conditions, minimizing the enzyme loss during co-precipitation. 31 The Ca-MOMs are also stable under both weakly acidic and basic conditions, 31 allowing for biocatalysis under the optimal pH of the immobilized enzyme.…”

“…Metal–organic frameworks (MOFs) are advanced platforms for enzyme immobilization and have provided advancement in biocatalysis, biomedicine, and fundamental biophysics research. − Thus far, major efforts in enzyme@MOF research have been focused on optimizing the metal, ligand, aperture, and/or pores of MOFs to enhance enzyme protection (against the reaction medium), reusability (as a result of the ease of separation), and substrate selectivity and/or diffusivity. − , While preformed, highly stable, and crystalline MOFs are mostly applied to host relatively small enzymes with small substrates, “one-pot” synthesis via co-crystallization of large enzymes and/or enzyme clusters with metals and ligands in the aqueous phase (also known as biomimetic mineralization) has been shown to be effective in removing the size limitation of enzymes. ,− Recently, researchers including our team have shown the possibility of using co-precipitation to remove the size limitation of the substrate, so that the entrapped enzymes are partially exposed to the reaction medium and partially buried under the MOF crystal surfaces, − as demonstrated on zeolitic imidazolate frameworks (ZIFs) and recent Ca-based metal–organic materials (MOMs, an analogue of MOFs but with a one- or two-dimensional structure); ,− the exposed enzyme regions were also revealed using our developed biophysical tools. , Importantly, our enzyme@Ca-MOM composites can be formed in the enzyme-friendly, aqueous phase under ambient conditions, minimizing the enzyme loss during co-precipitation . The Ca-MOMs are also stable under both weakly acidic and basic conditions, allowing for biocatalysis under the optimal pH of the immobilized enzyme.…”

Unveiling the orientation and dynamics of enzymes in unstructured artificial compartments of metal–organic frameworks (MOFs)

“…Such a unique enzyme immobilization approach has been observed and confirmed in several of our recent works. 21,[30][31][32][33][34][35][36] In this communication, we report the case of aq-ZIF. Because urea can only unfold the exposed portion of enzymes (the buried portion is protected by the MOF scaffolds), 21,44 column 3 of Table 1 reports the chance for different lys regions to be exposed (13-32% depending on labelled regions).…”

“…16,21,30 We found that the enzyme was confined both on the crystal surface ( partially embedded) and within crystal defects, a condition also discovered in our recent works. 21,[30][31][32][33][34][35][36] We then distinguished the latter from the former using a recently reported urea-perturbation strategy, 21 followed by revealing the backbone dynamics and contact residues of lys in the co-crystal defects. To our best knowledge, this is the first report on experimental unveiling of enzyme dynamics in unstructured artificial compartments.…”

Metal–Organic Materials (MOMs) Enhance Proteolytic Selectivity, Efficiency, and Reusability of Trypsin: A Time-Resolved Study on Proteolysis

“…Here, we combine MOMs, SDSL-EPR, and MS to overcome both challenges. Metal–organic frameworks (MOFs) are advanced platforms to immobilize enzymes. − We recently found a unique approach to immobilize enzymes with large substrates (the size of a protein or larger) on porous solid supports based on the cocrystallization of enzyme and certain metals/ligands in the enzyme-friendly, aqueous phase (forming the enzyme@MOMs composites). , A portion of the enzymes can be exposed above the MOM crystal surface to contact large substrates, which cannot diffuse into MOM pores (∼nm or smaller). − The buried portion allows for substantial protection against harsh environments including proteolytic conditions . SDSL-EPR is sensitive to molecular rotational tumbling rates and, thus, molecular size, as well as the population of the labeled peptides, regardless of the complexity of the studied system (protease and most MOMs do not influence EPR signal). − Thus, the molecular size and population of product polypeptides (if spin labeled on the truncation) can be monitored in real time by SDSL-EPR.…”