Enzyme immobilization in metal–organic frameworks (MOFs) offers retained enzyme integrity and activity, enhanced stability, and reduced leaching. Trapping enzymes on MOF surfaces would allow for catalysis involving large substrates. In both cases, the catalytic efficiency and selectivity depend not only on enzyme integrity/concentration but also orientation. However, it has been a challenge to determine the orientation of enzymes that are supported on solid matrices, which is even more challenging for enzymes immobilized/trapped in MOFs due to the interferences of the MOF background signals. To address such challenge, we demonstrate in this work the utilization of site-directed spin labeling in combination with Electron Paramagnetic Resonance spectroscopy, which allows for the first time the characterization of the orientation of enzymes trapped on MOF surfaces. The obtained insights are fundamentally important for MOF-based enzyme immobilization design and understanding enzyme orientation once trapped in solid matrices or even cellular confinement conditions.

By virtue of the atomic resolution of the SDSL-EPR technique and the on-demand COF syntheses, we show unambiguously that the degree of freedom of the encapsulated enzymes inside the nanopore varied along with the confined spatial environments. Increasing the hydrophilicity in the isoreticular COFs resulted in the accommodated enzyme with decreasing degrees of freedom and, consequently, lower reactivity. The developed structure-activity relationships are expected to be leveraged to tailor host materials for achieving more efficient formulations.

Gold nanoparticles (AuNPs) are advancing a number of areas. Meanwhile, the inevitable contact of AuNPs with surrounding biomolecules has been realized to “negatively” impact the nanoparticle (NP) function, biological environment, and even public health. Understanding the mechanisms of the NP–biomolecule interaction often leads to revealing the pathways toward the negative, deleterious effects. It is therefore essential to know the undesirable pathways so that these unwanted effects can be reduced by blocking or rerouting these pathways. A fundamental step to understand the interaction mechanism is to elucidate the changes in protein structure and dynamics and in NP properties upon the contact of AuNPs and protein, at the molecular level. However, it is challenging to probe these areas experimentally due to the large size and the complexity of the protein–nano interface. Here we report the use of a unique approach, electron paramagnetic resonance (EPR) spectroscopy, to probe the structure and dynamics of a model protein, T4 lysozyme (T4L), upon interaction with AuNPs. In combination with circular dichroism (CD) spectroscopy, UV–vis spectroscopy, and a protein activity assay, we found that T4L triggered the AuNPs to aggregate but remained in its native structure. Remarkably, our results also revealed the pathways of how T4L triggers AuNP aggregation, the potential applications of which were discussed. Lastly, this work demonstrated the usefulness of the EPR spectroscopy in probing the complexes formed by nanomaterials and macrobiomolecules, opening a new window to probe the nano–bio interface in the native state.

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Although enzyme immobilization has improved many areas, biocatalysis involving large-size substrates is still challenging for immobilization platform design because of the protein damage under the often “harsh” reaction conditions required for these reactions. Our recent efforts indicate the potential of using Metal–Organic Frameworks (MOFs) to partially confine enzymes on the surface of MOF-based composites while offering sufficient substrate contact. Still, improvements are required to expand the feasible pH range and the efficiency of contacting substrates. In this contribution, we discovered that Zeolitic Imidazolate Framework (ZIF) and a new calcium-carboxylate based MOF (CaBDC) can both be coprecipitated with a model large-substrate enzyme, lysozyme (lys), to anchor the enzyme on the surface of graphite oxide (GO). We observed lys activity against its native substrate, bacterial cell walls, indicating lys was confined on composite surface. Remarkably, lys@GO/CaBDC displayed a stronger catalytic efficiency at pH 6.2 as compared to pH 7.4, indicating CaBDC is a good candidate for biocatalysis under acidic conditions as compared to ZIFs which disassemble under pH < 7. Furthermore, to understand the regions of lys being exposed to the reaction medium, we carried out a site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy study. Our data showed a preferential orientation of lys in GO/ZIF composite, whereas a random orientation in GO/CaBDC. This is the first report on immobilizing solution-state large-substrate enzymes on GO surface using two different MOFs via one-pot synthesis. These platforms can be generalized to other large-substrate enzymes to carry out catalysis under the optimal buffer/pH conditions. The orientation of enzyme at the molecular level on composite surfaces is critical for guiding the rational design of new composites.