Comment on mr-2022-3

Abstract

Ligating a protein at a specific site with a tag molecule containing a paramagnetic metal ion provides a versatile way of generating pseudocontact shifts (PCS) in nuclear magnetic resonance (NMR) spectra. PCSs can be observed for nuclear spins far from the tagging site and PCSs generated from multiple tagging sites have been shown to enable highly accurate structure determinations. The present work investigates the situation, where only the local structure of a protein region or bound ligand is to be determined rather than the structure of the entire molecular system. In this case, the need for gathering structural information from tags deployed at multiple sites may be queried. Our study presents a computational simulation of the structural information available from samples produced with single tags attached at up to six different sites, up to six different tags attached to a single site, as well as scenarios in-between. The results indicate that the number of tags is much more important than the number of tagging sites. This has important practical implications, as it is much easier to identify a single site that is suitable for tagging than multiple ones. In an initial experimental demonstration with the ubiquitin mutant S57C, PCSs generated with four different tags at a single site are shown to accurately pinpoint the location of amide protons in different segments of the protein.