Preprints
https://doi.org/10.5194/mr-2021-63
https://doi.org/10.5194/mr-2021-63

  06 Oct 2021

06 Oct 2021

Review status: this preprint is currently under review for the journal MR.

Localising individual atoms of tryptophan side chains in the metallo-β-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites

Henry W. Orton1,, Iresha D. Herath2,, Ansis Maleckis3, Shereen Jabar2, Monika Szabo4, Bim Graham4, Colum Breen5, Lydia Topping5, Stephen J. Butler5, and Gottfried Otting1 Henry W. Orton et al.
  • 1ARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
  • 2Research School of Chemistry, The Australian National University, Sullivans Creek Road, Canberra ACT 2601, Australia
  • 3Latvian Institute of Organic Synthesis, Aizkraukles 21, LV-1006 Riga, Latvia
  • 4Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia
  • 5Department of Chemistry, Loughborough University, Epinal Way, Loughborough, LE11 3TU, United Kingdom
  • These authors contributed equally to this work.

Abstract. The metallo-β-lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal structures, which may assist in substrate binding and enzymatic activity. To probe the position of this loop, we labelled the tryptophan residues of IMP-1 with 7-13C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy (Δχ) tensors were determined by measuring pseudocontact shifts (PCS) of backbone amide protons. The Δχ tensors were subsequently used to identify the atomic coordinates of the tryptophan side chains in the protein. The PCSs were sufficient to determine the location of Trp28, which is located in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect was attributed to the shallow angle with which PCS isosurfaces tend to intersect if generated by tags and tagging sites that are identical except for the paramagnetic lanthanoid ion.

Henry W. Orton et al.

Status: open (until 03 Nov 2021)

Comment types: AC – author | RC – referee | CC – community | EC – editor | CEC – chief editor | : Report abuse
  • RC1: 'Comment on mr-2021-63', Marcellus Ubbink, 15 Oct 2021 reply

Henry W. Orton et al.

Henry W. Orton et al.

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Short summary
This manuscript describes a method for determining the solution structure of a solvent-exposed polypeptide segment (the L3 loop), which is next to the active site of the penicillin-degrading enzyme IMP-1. Tagging three different sites on the protein with paramagnetic metal ions allowed positioning the L3 loop with atomic resolution.