Improving the Accuracy of NMR Structures of Large Proteins Using Pseudocontact Shifts as Long-Range Restraints

Abstract

We demonstrate improved accuracy in protein structure determination for large (>/=30 kDa), deuterated proteins (e.g. STAT4(NT)) via the combination of pseudocontact shifts for amide and methyl protons with the available NOEs in methyl-protonated proteins. The improved accuracy is cross validated by Q-factors determined from residual dipolar couplings measured as a result of magnetic susceptibility alignment. The paramagnet is introduced via binding to thiol-reactive EDTA, and multiple sites can be serially engineered to obtain data from alternative orientations of the paramagnetic anisotropic susceptibility tensor. The technique is advantageous for systems where the target protein has strong interactions with known alignment media.

Extending the Size of Protein–RNA Complexes Studied by Nuclear Magnetic Resonance Spectroscopy

“…In this case, additional structural information can be obtained from pseudocontact shifts and residual dipolar couplings from magnetic alignment. [55][56][57] For the study of large protein-RNA complexes, spin-labeling of the RNA component in several locations along the RNA sequence might generate enough distance restraints to sufficiently define the location of the protein and RNA components. A useful spin label for RNA couples 3-(2-iodoacetamidoproxyl) to a 4-thiouridine base, [54] which does not appear to disturb the base-stacking pattern in double-stranded RNA.…”

Paramagnetic tagging of diamagnetic proteins for solution NMR

“…5 Another aspect is the study of the structure of symmetrical homodimers. 6 While external alignment can only reveal the axis of symmetry as one of the three principal axes of the alignment tensor, incomplete labelling of a homodimer with a paramagnetic tag leads to two sets of signals that contain information about the relative orientation and contact surfaces of the homodimer.…”

Paramagnetic Tools in Protein NMR

“…Commonly used are Mn 2 þ and Cu 2 þ for PREs and Co 2 þ , low-spin Fe 3 þ and Ni 2 þ for PCSs [37][38][39][40][41][42], either as naturally occurring cofactors in metal proteins or as artificial substitutes. Mn 2 þ , Co 2 þ and sometimes Cu 2 þ are also used in paramagnetic tags [43][44][45].…”