The resultant enzymatic performance depends on the orientation and dynamics of the entrapped enzymes, which are often challenging to determine at the sufficiently high resolution, due to the interference of the porous background ( Pan et al., 2021a). We recently demonstrated a unique approach to bridge this knowledge gap using site-directed spin labeling (SDSL) in combination with Electron Paramagnetic Resonance (EPR) spectroscopy ( Pan et al., 2021a). SDSL-EPR has been proved powerful in determining protein and other biomacromolecule structural information in complex biological systems our team recently extended it to the interface of enzyme and mesoporous materials, which led to meaningful structural information that explains the resultant enzyme activity ( Pan et al., 2021b, Sun et al., 2019, Pan et al., 2018). Thus far, due to the uniqueness of EPR spectroscopy especially spectral simulation, there has been limited step-by-step procedure reported for utilizing SDSL-EPR on probing structural information at the enzyme-porous materials interface. Therefore, here we present a detailed protocol for using our strategy to fill in this gap. We particularly focus on determining the relative orientation of enzyme in respect to the pore surfaces as well as the backbone dynamics of enzyme upon encapsulation. The protocol was tested on a model enzyme, lysozyme, upon encapsulation in Metal-Organic Frameworks/Covalent-Organic Frameworks (MOFs/COFs). Lysozyme is a good model because both large (∼μm) and small (∼nm) size substrates can be catalyzed by this enzyme ( Vocadlo et al., 2001, Kao et al., 2014). Meanwhile, MOFs/COFs are advanced porous platforms for enzyme encapsulation ( Howarth et al., 2016, Majewski et al., 2017, Drout et al., 2019, Wang et al., 2020, Gkaniatsou et al., 2017, Lyu et al., 2014, Lian et al., 2017, Li et al., 2020, Farmakes et al., 2020, Neupane et al., 2019).

Note that, a unique feature of SDSL-EPR is that it is immune of the background matrices (under low water volume see below) ( Pan et al., 2021a). Thus, with minimal modifications, the protocol can be used to determine the orientation and dynamics of other enzymes upon entrapping in other porous materials. As is evident from the name, SDSL-EPR requires both protein spin labeling and EPR measurement.

Therefore, in this protocol, we strive our best to cover as many details as possible in both aspects, so that the protocol can be generalized to other enzymes/materials. Before one begins, the following Preparations need to be carried out.Īcquire/collect available structural information of the spin label on proteins. This step is necessary because a spin label is essentially a protein sidechain.

Bruker epr iris motor