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Biosynthetic production and evaluation of knotted peptide topology and characteristics.

Babu, Charles, Phulera, Swastik, Hao, Qin, Wall, Daniel, Nettleton, David, Pathak, Tejas and Siuti, Piro (2021) Biosynthetic production and evaluation of knotted peptide topology and characteristics. ACS Omega, 6 (44). pp. 29555-29566. ISSN 2470-13432470-1343


Knotted peptides present a wealth of structurally diverse, biologically-active molecules, with the inhibi-tor cystine knot or knottin folds among the most prevalent. Many of these natural products interact with extracellular targets such as voltage-gated ion channels with exquisite selectivity and potency, making them intriguing therapeutic modalities. However, such compounds are often produced by exotic organisms in low concentrations, making structure determination and biological characterization challenging. Heterologous expression in bacterial hosts could potential-ly solve these issues by making scalable production of these compounds accessible - though this methodology would rely on correct in vivo disulfide formation and folding in the absence of native oxidative folding pathways that are pre-sent in the original organisms. We screened expression constructs for a heterologously biosynthesized knotted peptide to determine the most influential parameters for successful disulfide folding using NMR spectroscopic fingerprinting to validate the topological structure of folded products. To better understand this emerging modality of peptides, we performed pharmacokinetic characterization which indicate the interlocking disulfide structure minimizes liabilities of linear peptide sequences and has a profound influence on these molecules’ behavior in vivo. We then developed an assay to study the solution folding of toxin residues in real time, providing a method for studying the complex folding process these molecules undergo during maturation.

Item Type: Article
Date Deposited: 07 Dec 2021 00:45
Last Modified: 07 Dec 2021 00:45