Enzymatic thioamidation of peptide backbones

Andi Liu, P. H. Krushnamurthy, K. S. Subramanya, Douglas A. Mitchell, Nilkamal Mahanta

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Thioamides are found in a few natural products and two known protein assemblies: the Escherichia coli ribosome and methyl-coenzyme M reductase (MCR) from methane-metabolizing archaea. Compared to an amide, thioamides alter the physical and chemical properties of peptide backbones, including the conformation dynamics, proteolytic stability, hydrogen-bonding capabilities, and possibly reactivity of a protein when installed. Recently, there has been significant progress in elucidating enzymatic post-translational thioamide installation, with most work leveraging the archaeal MCR-modifying enzymes. This chapter describes the protocols used for the in vitro enzymatic thioamidation of MCR-derived peptides, including polypeptide overexpression, purification, reaction reconstitution, and mass spectrometry-based product analysis. In addition, we highlight the protocols used for the biochemical, kinetics, and binding studies using recombinant enzymes obtained heterologously from E. coli. We anticipate that these methods will serve to guide future studies on peptide post-translational thioamidation, as well as other peptide backbone modifications using a parallel workflow.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
DOIs
StateAccepted/In press - 2021

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • ATP
  • Fluorescence polarization
  • MCR
  • O-phosphorylation
  • PTM
  • Recombinant proteins
  • TfuA
  • Thioamidation
  • YcaO

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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