Increasing the Chemical‐Shift Dispersion of Unstructured Proteins with a Covalent Lanthanide Shift Reagent

Abstract

The study of intrinsically disordered proteins (IDPs) by NMR often suffers from highly overlapped resonances that prevent unambiguous chemical-shift assignments,a nd data analysis that relies on well-separated resonances.W ep resent ac ovalent paramagnetic lanthanide-binding tag (LBT) for increasing the chemical-shift dispersion and facilitating the chemical-shift assignment of challenging, repeat-containing IDPs.Linkage of the DOTA-based LBT to ac ysteine residue induces pseudo-contact shifts (PCS) for resonances more than 20 residues from the spin-labeling site.This leads to increased chemical-shift dispersion and decreased signal overlap,thereby greatly facilitating chemical-shift assignment. This approachis applicable to IDPs of varying sizes and complexity,a nd is particularly helpful for repeat-containing IDPs and lowcomplexity regions.T his results in improved efficiency for IDP analysis and binding studies.The well-established structure-function paradigm has been challenged by the discovery of intrinsically disordered functional proteins (IDPs).[1] Although lacking stable secondary or tertiary structure elements,this large class of proteins plays ac rucial role in various cellular processes.[2] It is suggested that about 40 %ofall proteins have disordered regions of 40 or more residues,w ith many proteins existing solely in the unfolded state.[3]NMR spectroscopy is aw ell-suited method for studying the residual structure,dynamics,and interactions of IDPs with atomic resolution under near-native conditions.[4] Thek ey step for these studies is the assignment of NMR resonances, which is particularly challenging for IDPs owing to poor chemical-shift dispersion, which results in severe spectral overlap.T his is due to alack of well-defined hydrogen bonds and am issing hydrophobic core with aromatic contributions.[5] To reduce spectral overlap,s everal approaches have been proposed. In the "divide and conquer" approach, chemical-shift assignments of shorter constructs obtained by standard NMR experiments are transferred to the full-length protein.[6] To assign chemical shifts of full-length unstructured proteins,e xperiments have been proposed that make use of the favorable relaxation properties of IDPs in high-dimensional experiments (up to 7D). [7] These experiments reduce resonance overlap and can potentially provide unambiguous chemical-shift assignments.A na lternative strategy is the direct detection of heteronuclei, using the beneficial chemical-shift dispersion of 13 C [8] or the increased resolution of direct detection of 15 N. [9] These approaches can be implemented easily with nonuniform sampling combined with nonFourier signal processing methods and fast-pulsing techniques in order to reduce measurement time. [10] Nevertheless,even providing that complete chemical-shift assignments can be obtained, missing chemical-shift dispersion in 2D NMR experiments complicates standard NMR approaches,including chemical-shift perturbation mapping of protein-ligand interactions and studies of protein ...

Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation

“…In support of the second hypothesis, namely that TNPO1 interferes with phase separation of FUS by interacting with arginines in the C-terminal RGG3-PY domain, we have previously shown that TNPO1 directly binds to a synthetic FUS-RGG3 peptide (Dormann et al, 2012) and to several arginine residues in the FUS-RGG3 region (R472, R473, R476) (Gö bl et al, 2016). This interaction appears to be mainly charge-driven, as we observed a strong decrease in binding of TNPO1 to RGG3-PY at higher salt concentration ( Figure S4A and Table S1).…”

Chiral DOTA chelators as an improved platform for biomedical imaging and therapy applications

“…Studies with different Ln(III) ions also revealed no change in the isomer ratios, dismissing the influence of metal ion size. Besides MRI, the control of the stereoisomerism of the complexes is also very important for some other applications: The stereoisomeric purity of chiral luminescent lanthanide complexes are crucial for circularly polarised luminescence (CPL) applications 30 , 40 ; Such rigid paramagnetic complexes are also ideal pseudocontact shift tags for proteins for NMR studies in solution and even in cells 41 , 42 .…”

Methyl group reorientation under ligand binding probed by pseudocontact shifts

“…A straightforward application of paramagnetic tagging is the additional dispersion induced by PCS. Paramagnetic tags have already been used to disperse resonances of an intrinsically disordered protein, presenting a crowded amide proton spectrum (Gobl et al 2016 ). Our data demonstrate again that additional dispersion is also relevant for folded, larger proteins that are being studied with methyl group NMR probes, because two-dimensional spectra usually show considerable crowding for methyl resonances in the 1 H region of 0.8–1.0 ppm, as was reported and used before (Sattler and Fesik 1997 ).…”