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Enzyme encapsulation in metal-organic frameworks (MOFs)/covalent-organic frameworks (COFs) provides advancement in biocatalysis, yet the structural basis underlying the catalytic performance is challenging to probe. Here, we present an effective protocol to determine the orientation and dynamics of enzymes in MOFs/COFs using site-directed spin labeling and electron paramagnetic resonance spectroscopy. The protocol is demonstrated using lysozyme and can be generalized to other enzymes. 

Protein transfer into nanoscale compartments is critical for many cellular/life processes, yet there are few reports on how compartment properties impact the protein orientation during a transfer. Such a knowledge gap limits a deeper understanding of the protein transfer mechanism, which could be bridged using nanoporous materials. Here, we use a mesoporous silica, a covalent organic framework, and a metal-organic framework with charged, hydrophobic, and neutral surfaces, respectively, to elucidate the impact of channel properties on the transfer of a model protein, lysozyme. Using site-directed spin labeling and time-resolved electron paramagnetic resonance spectroscopy, we reveal that the transfer can be a multi-step process depending on channel properties and depict the relative orientation changes of lysozyme upon transfer into each channel. To the best of our knowledge, this is the first structural insight into protein orientation upon transfer into different compartments, meaningful for the rational design of synthetic materials to host enzymes or mimic the cellular compartments.