Poster Presentation International Peptide Symposium 2023

Chemically Modified Amino Acids as Site-Specific 19F-NMR Probes in Proteins (#367)

Yi Jiun Tan 1 2 , Haocheng Qianzhu 2 , Elwy Abdelkader Ali 1 2 , Iresha Herath 2 , Thomas Huber 2 , Gottfried Otting 1 2
  1. ARC Centre of Excellence for Innovations in Peptide & Protein Science, RSC, ANU, Canberra, ACT, Australia
  2. RSC, ANU, Canberra, ACT, Australia

Fluorine atoms are known to display scalar 19F−19F couplings in nuclear magnetic resonance (NMR) spectra when they are sufficiently close in space for nonbonding orbitals to overlap. Specifically, through-space scalar 19F–19F (TSJFF) couplings can be observed between fluorinated noncanonical amino acids positioned in the hydrophobic core of a protein or between CF3 groups of solvent exposed residues (Orton et al., 2021). Using N6-(trifluoroacetyl)-L-lysine (TFA-Lys), we show that 19F−19F TOCSY peaks can also be observed between two TFA-Lys residues site-specifically incorporated into AncCDT-1, which is a two-domain Arg-binding protein assuming a closed conformation in the presence of Arg and an open conformation in the free state. TFA-Lys can readily be installed in proteins in response to an amber stop codon (one in each domain) so that 19F−19F contacts between the TFA-Lys residues signal the closed conformation. In addition, TFA-Lys was site-specifically incorporated into the MARCKS peptide at position S8 using the calmodulin fusion tag system (Ishida et al., 2016). The incorporation efficiency was practically complete, and 3 mg purified peptide could be obtained from 1 L of E. coli cell culture. The system also provided high yields (15–22 mg/L) of 15N-labelled MARCKS peptide when expression was conducted in a bioreactor. The system works much better for the production of intrinsically disordered peptides than any other fusion system we have tested.

  1. Ishida, H., Nguyen, L. T., Gopal, R., Aizawa, T., Vogel, H. J. 2016. Overexpression of antimicrobial, anticancer, and transmembrane peptides in Escherichia coli through a calmodulin-peptide fusion system. J. Am. Chem. Soc. 138, 11318-11326.
  2. Orton, H. W., Qianzhu, H., Abdelkader, E. H., Habel, E. I., Tan, Y. J., Frkic, R. L., Jackson, C. J., Huber, T., Otting, G. 2021. Through-space scalar 19F–19F couplings between fluorinated noncanonical amino acids for the detection of specific contacts in proteins. J. Am. Chem. Soc. 143, 19587–19598.