Metal-Organic Materials (MOMs) Enhance Proteolytic Selectivity, Efficiency, and Reusability of Trypsin: A Time-Resolved Study on Proteolysis

Qiaobin Li; Zoe Armstrong; Austin MacRae; Angel Ugrinov; Li Feng; Bingcan Chen; Ying Huang; Hui Li; Yanxiong Pan; Zhongyu Yang

doi:10.1021/acsami.2c19873

Metal-Organic Materials (MOMs) Enhance Proteolytic Selectivity, Efficiency, and Reusability of Trypsin: A Time-Resolved Study on Proteolysis

ACS Appl Mater Interfaces. 2023 Feb 9. doi: 10.1021/acsami.2c19873. Online ahead of print.

Authors

Affiliations

¹ Department of Chemistry and Biochemistry, North Dakota State University, Fargo, North Dakota 58102, United States.
² Department of Plant Sciences, North Dakota State University, Fargo, North Dakota 58102, United States.
³ Department of Civil, Construction, and Environmental Engineering, North Dakota State University, Fargo, North Dakota 58102, United States.
⁴ State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Changchun 130022, China.

PMID: 36757369
DOI: 10.1021/acsami.2c19873

Abstract

Proteases are involved in essential biological functions in nature and have become drug targets recently. In spite of the promising progress, two challenges, (i) the intrinsic instability and (ii) the difficulty in monitoring the catalytic process in real time, still hinder the further understanding and engineering of protease functionalities. These challenges are caused by the lack of proper materials/approaches to stabilize proteases and monitor proteolytic products (truncated polypeptides) in real time in a highly heterogeneous reaction mixture. This work combines metal-organic materials (MOMs), site-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy, and mass spectrometry (MS) to overcome both barriers. A model protease, trypsin, which cleaves the peptide bonds at lysine or arginine residues, was immobilized on a Ca-MOM via aqueous-phase, one-pot cocrystallization, which allows for trypsin protection and ease of separation from its proteolytic products. Time-resolved EPR and MS were employed to monitor the populations, rotational motion, and sequences of the cleaved peptide truncations of a model protein substrate as the reaction proceeded. Our data suggest a significant (at least 5-10 times) enhancement in the catalytic efficiency (k_cat/k_m) of trypsin@Ca-MOM and excellent reusability as compared to free trypsin in solution. Surprisingly, entrapping trypsin in Ca-MOMs results in cleavage site/region selectivity against the protein substrate, as compared to the near nonselective cleavage of all lysine and arginine residues of the substrate in solution. Remarkably, immobilizing trypsin allows for the separation and, thus, MS study on the sequences of truncated peptides in real time, leading to a time-resolved "movie" of trypsin proteolysis. This work demonstrates the use of MOMs and cocrystallization to enhance the selectivity, catalytic efficiency, and stability of trypsin, suggesting the possibility of tuning the catalytic performance of a general protease using MOMs.

Keywords: SDSL-EPR; metal−organic materials; proteolytic efficiency; proteolytic selectivity; tandem MS.