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

Abstract

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy probes the otherwise inaccessible structural information in complex biological systems. We recently extended SDSL-EPR to reveal the relative orientation and backbone dynamics of enzymes upon encapsulation in mesoporous nanostructures, which set the structural basis underlying the observed biocatalytic activity. Our strategy had generated interest in the biocatalysis community, and thus in this resource article, we contribute an introduction to the principles and experimental procedure that generalize SDSL-EPR to heterogeneous biocatalysis. We will focus on enzymes in mesoporous materials with examples demonstrating the methods and cautions of potential pitfalls. The ultimate goal is to provide the biocatalysis community with a powerful resource to fill in a long-standing knowledge gap in heterogeneous biocatalysis and the structure-function relationship of enzymes at the interface of enzyme-mesoporous materials and utilize the structural insights to guide the rational design of porous platforms for enzyme immobilization. • Enzyme orientation is determined by using EPR to probe its contact with nanoscale pores • Enzyme dynamics in nanoscale pores and associated catalytic activities are revealed • Protocols and procedures are given for experimental data acquisition, analysis, and interpretation • Various EPR techniques and spin labels can be extended to heterogeneous biocatalysis Enzyme immobilization in nanoscale confinement offers enhanced enzyme protection and reusability, yet the structure-function relationship of the entrapped enzymes remains elusive. Site-directed spin labeling in combination with electron paramagnetic resonance (EPR) spectroscopy has primarily been applied in determining protein structural information in complex biological systems. In this resource article, we introduce the extension of the X-band continuous-wave EPR with a methanethiosulfonate spin label to reveal enzyme structural basis upon immobilization into nanoscale confinement. Using our recent works as examples, we demonstrate the method for determining enzyme orientation and backbone dynamics upon immobilization with a special focus on generalizing the method to heterogeneous biocatalysis and cautions in each step. Furthermore, we summarize the recent findings in other EPR techniques and spin labels so that the whole EPR toolbox can be extended to heterogeneous biocatalysis. In this resource article, Yang and colleagues introduce a general procedure that can reveal the orientation and backbone dynamics of enzymes entrapped in mesoporous materials based on metal-organic materials (MOMs) by using X-band SDSL-EPR. They demonstrate the method by using recently published works focusing on enzyme-MOM interfaces, as well as highlight cautions and potential solutions at each step. They then summarize a variety of other EPR techniques and spin labels in order to pinpoint their potential applications in heterogeneous biocatalysis in the near future.