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Biophysical and Functional Characterization of a Thermally Stable Bifunctional Serine Protease Inhibitor from Cleome viscosa Seeds

Year: 2025

Type: article

Abstract: Plant protease inhibitors (PPI) play a significant role against microbes, insects, and, to a considerable extent, human pathogens. PPIs inactivate hydrolase enzymes or depolarize the plasma membrane of the pathogens, thereby inhibiting their growth, replication, and invasion. Here, an active serine protease inhibitor was isolated and purified from the seeds of Cleome viscosa. The purified inhibitor was homogenous and exhibited a molecular weight of around 12 kDa as a monomer. The secondary structure analysis indicated that the inhibitor was predominantly composed of α-helical content. The kinetics experiments demonstrated a noncompetitive mode of inhibition towards serine protease when casein was used as the enzyme substrate. The inhibitor formed a stable complex with serine protease, having a likely 1:1 stoichiometry, as inferred from ITC, and the dissociation constant was examined to be Kd = 1.9 × 10−6 M with a Gibbs free energy of ΔG = −8.079 (kcal/mol). The inhibitor exhibits stable protease inhibition up to 90 °C. Further, in vitro preliminary studies revealed its inhibitory effects against HSV-2 function, evidence that it may have a role in the treatment of viral infections.

Source: International Journal of Molecular Sciences

DOI: 10.3390/ijms262411792

Authors: Manohar Radhakrishnan, Vajaravijayan Senthilvadivelu, Eswar Kumar Nadendla, Kundan Sivashanmugan, Gunasekaran Krishnasamy

Institutions: University of Madras, Indiana University Indianapolis, Indiana University School of Medicine, St. Jude Children's Research Hospital, University of Maryland, Baltimore

Language: English

Topic: Protease and Inhibitor Mechanisms

Subfield: Cancer Research

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,499

Funder: Central Leather Research Institute