Protocol for resolving enzyme orientation and dynamics in advanced porous materials via SDSL-EPR

• Published in: STAR protocols Vol. 2; no. 3; p. 100676
• Main Authors: Pan, Yanxiong, Li, Hui, Li, Qiaobin, Lenertz, Mary, Schuster, Isabelle, Jordahl, Drew, Zhu, Xiao, Chen, Bingcan, Yang, Zhongyu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.09.2021; Elsevier
• Subjects: Biophysics; Chemistry; Material sciences; Molecular/Chemical Probes; Protein Biochemistry; Protein expression and purification; Protocol; Structural Biology; Protein Biochemistry; Chemistry; Protein expression and purification; Structural Biology; Material sciences; Molecular/Chemical Probes; Biophysics
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2021.100676

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. For complete information on the generation and use of this protocol, please refer to Pan et al. (2021a). [Display omitted] •A protocol to resolve protein orientation/dynamics in porous materials is provided•Site-directed spin labeling is combined with electron paramagnetic resonance•Principles of protein labeling and key data acquisition steps are summarized•Spectral simulation details with troubleshooting procedures are detailed 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.