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

The metallo-ß-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 (PCSs) 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 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.

Localising nuclear spins by pseudocontact shifts from a single tagging site.

Ligating a protein at a specific site with a tag molecule containing a paramagnetic metal ion provides a versatile way of generating pseudocontact shifts (PCSs) 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 at specific sites of interest, even when using flexible tags, provided the fitted effective magnetic susceptibility anisotropy (Δχ) tensors accurately back-calculate the experimental PCSs measured in the immediate vicinity of the site of interest. 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, and in-between scenarios. The results indicate that the number of tags is 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.