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

Abstract

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 (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

“…Shallow intersection angles are commonly observed when the same tag loaded with a different paramagnetic lanthanoid ion (such as Tb versus Tm ) is deployed at the same tagging site. Previous work, where we used the PCSs from three different tagging sites to determine the location of a solvent-exposed protein loop, showed that the localisation coordinates determined by PCSs were more accurate when all data encompassed PCSs measured with only a single paramagnetic metal ion . Therefore, we conducted the experiments with ubiquitin using Tm tags only.…”

“…In the case of the ubiquitin data used in this work, the uncertainty of the PCS data associated with the amide protons of segments A–C was small (estimated to be less than 0.01 ppm). A straightforward approach that indirectly captures the effect of most of the sources of uncertainty on the localisation spaces relies on performing the tensor fits multiple times with random omission of a certain percentage of the PCSs .…”

Atomistic Simulations for Reactions and Vibrational Spectroscopy in the Era of Machine Learning─Quo Vadis?

“…This is largely due to the possibility to explicitly follow the dynamics of molecular systems with the full information about observables contained in the time series of positions and velocities within the framework of classical dynamics. The “translation” between the output from such a molecular dynamics (MD) simulation (positions and velocities as a function of time) is accomplished by virtue of statistical mechanics, in particular the correlation function formalism developed and introduced in the 1960s. − …”

Localising nuclear spins by pseudocontact shifts from a single tagging site

“…The orientation of an effective ∆χ tensor is predominantly governed by the steric and chemical environment of the tag and therefore difficult to predict. It has been observed many times, however, that different paramagnetic metal ions generally produce very similar ∆χ-tensor orientations, if the chemical structure of the tag and tagging site are the same (Su et al, 2008;Keizers et al, 2008;Man et al, 2010;Graham et al, 2011;Joss et al, 2018;Zimmermann et al, 2019;Orton et al, 2022). Similar ∆χ-tensor orientations lead to shallow intersection angles of the respective PCS isosurfaces.…”

“…Shallow intersection angles are commonly observed when the same tag loaded with a different paramagnetic lanthanoid ion (such as Tb 3+ versus Tm 3+ ) is deployed at the same tagging site. Previous work, where we used the PCSs from three different tagging sites to determine the location of a solvent-exposed protein loop, showed that the localisation coordinates determined by PCSs were more accurate, when all data encompassed PCSs measured with only a single paramagnetic metal ion (Orton et al, 2022). Therefore, we conducted the experiments with ubiquitin using Tm 3+ tags only.…”