Paramagnetic relaxation enhancement to improve sensitivity of fast NMR methods: application to intrinsically disordered proteins

Abstract

We report enhanced sensitivity NMR measurements of intrinsically disordered proteins in the presence of paramagnetic relaxation enhancement (PRE) agents such as Ni(2+)-chelated DO2A. In proton-detected (1)H-(15)N SOFAST-HMQC and carbon-detected (H-flip)(13)CO-(15)N experiments, faster longitudinal relaxation enables the usage of even shorter interscan delays. This results in higher NMR signal intensities per units of experimental time, without adverse line broadening effects. At 40 mmol·L(-1) of the PRE agent, we obtain a 1.7- to 1.9-fold larger signal to noise (S/N) for the respective 2D NMR experiments. High solvent accessibility of intrinsically disordered protein (IDP) residues renders this class of proteins particularly amenable to the outlined approach.

19F Paramagnetic Relaxation Enhancement: A Valuable Tool for Distance Measurements in Proteins

“…In cases where NOE data were found limited, distances derived from PREs provided valuable supplemental restraints and permitted either characterization of the global folds for some proteins, [1,2] or delineate structural properties of disorder proteins. [3,4] Correspondence to: Angela M. Gronenborn, amg100@pitt.edu. Supporting information for this article is given via a link at the end of the document.…”

Low concentration of a Gd-chelate increases the signal-to-noise ratio in fast pulsing BEST experiments

“…Ultrafast NMR can be combined with SOFAST-HMQC 2D experiments into a single, relaxation optimized and spatially encoded tool [21]. During the preparation of this manuscript, another group reported that the addition of a paramagnetic nickel chelate improved the signal to noise ratio of SOFAST-HMQC experiments recorded for unfolded proteins [37]. Here, we show that 2D and 3D BEST sequences benefit from the presence of small concentrations of a gadolinium chelate for several different proteins, including both folded and unfolded examples.…”

“…Gadolinium may be preferred to nickel chelate due to its lower concentration requirements, 1 mM and 50 mM, respectively. Although nickel chelate has proven to enhance signal sensitivity for unfolded proteins using SOFAST-HMQC experiments [37], the pioneer work performed by Cai and collegues [23] seems to indicate that this effect is dependent of the water return to equilibrium. Indeed, their work suggests that for experiments with water flip back pulses, residues exposed to solvent experience signal reduction when fast pulsing is combined with Ni(DO2A).…”

“…Recently it has been shown that addition of the Ni 2+ -chelated paramagnetic agent (1,4,7,10-tetraazacyclododecane-1,7-bis(acetic acid) or DO2A) significantly improves the signal-to-noise ratio of disordered proteins [37] using another fast pulsing sequence, the so-called SOFAST-HMQC [19]. This approach developed in Selenko's group was based on the fact that the high solvent exposure of intrinsically disordered proteins (IDP) residues ensures an uniform T 1 shortening along the sequence.…”

Nuclear spin relaxation in liquids and gases

“…153 The PRE may also be used for other than structural purposes. Theillet et al 154 proposed the use of PRE to improve sensitivity of fast NMR methods, illustrated by the case of intrinsically disordered proteins, in the presence of Ni(II) chelates. Saio and co-workers 155 described an NMR strategy for fragment-based ligand screeining using paramagnetic lanthanide probes.…”