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Enzymatic thioamidation of peptide backbones
Andi Liu1, P H Krushnamurthy2, K S Subramanya2
1Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana, IL, United States; Department of Microbiology, University of Illinois, Urbana, IL, United States.
Methods in Enzymology
|July 30, 2021
Summary
This study details methods for in vitro enzymatic thioamidation of peptides, crucial for understanding protein modifications. These protocols aid research into thioamide installation and other peptide backbone changes.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Thioamides, found in natural products and protein assemblies like MCR, modify peptide backbone properties.
- Enzymatic post-translational thioamide installation is an emerging area, primarily studied using archaeal MCR-modifying enzymes.
Purpose of the Study:
- To describe established protocols for in vitro enzymatic thioamidation of MCR-derived peptides.
- To present methods for biochemical, kinetics, and binding studies of recombinant enzymes.
- To provide a framework for future research on peptide backbone modifications.
Main Methods:
- Polypeptide overexpression and purification.
- In vitro reaction reconstitution for thioamidation.
- Mass spectrometry for product analysis.
- Heterologous expression of recombinant enzymes in E. coli.
Main Results:
- Established protocols for enzymatic thioamidation of MCR-derived peptides.
- Detailed methods for biochemical and binding studies using recombinant enzymes.
- Demonstrated a parallel workflow applicable to various peptide modifications.
Conclusions:
- The described methods facilitate in vitro enzymatic thioamidation of peptides.
- These protocols support the study of thioamide installation and other peptide backbone modifications.
- The workflow provides a foundation for future investigations in protein chemistry.
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