Sensitivity enhancement using paramagnetic relaxation in MAS solid-state NMR of perdeuterated proteins

Abstract

Previously, Ishii et al., could show that chelated paramagnetic ions can be employed to significantly decrease the recycle delay of a MAS solid-state NMR experiment [N.P. Wickramasinghe, M. Kotecha, A. Samoson, J. Past, Y. Ishii, Sensitivity enhancement in C-13 solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing H-1 T-1 relaxation, J. Magn. Reson. 184 (2007) 350-356]. Application of the method is limited to very robust samples, for which sample stability is not compromised by RF induced heating. In addition, probe integrity might be perturbed in standard MAS PRE experiments due to the use of very short duty cycles. We show that these deleterious effects can be avoided if perdeuterated proteins are employed that have been re-crystallized from D 2 O:H 2 O = 9:1 containing buffer solutions. The experiments are demonstrated using the SH3 domain of chicken a-spectrin as a model system. The labeling scheme allows to record proton detected 1 H, 15 N correlation spectra with very high resolution in the absence of heteronuclear dipolar decoupling. Cu-edta as a doping reagent yields a reduction of the recycle delay by up to a factor of 15. In particular, we find that the 1 H T 1 for the bulk H N magnetization is reduced from 4.4 s to 0.3 s if the Cu-edta concentration is increased from 0 mM to 250 mM. Possible perturbations like chemical shift changes or line broadening due to the paramagnetic chelate complex are minimal. No degradation of our samples was observed in the course of the experiments.

Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure

“…To exploit PRE (Paramagnetic Relaxation Enhancement), Cu(edta) was added to the monomeric Ab 1-40 peptide, prior to fibrilization, at a concentration of 75 mM. 40,41 Growth and quality of the fibrils were monitored using EM. For each sample, typically 10 mg of Ab 1-40 fibrils were packed into a 3.2 mm MAS solid-state NMR rotor.…”

“…These are based on MAS solidstate NMR experiments, [5][6][7][8][9][10][11] and cryo-electron microscopic image reconstructions. 12 Even though there is some controversy concerning the conformational space that Ab fibrils can adopt, all published models, including the 2-fold 7,10 and 3-fold 9 symmetric Ab 1-40 fibril structure suggested by Tycko, and Bertini and co-workers, agree on the basic building block which involves a b-sheet (b1, residues 12-24), a turn, and a second b-sheet (b2, residues [28][29][30][31][32][33][34][35][36][37][38][39][40]. Biophysical studies indicate a certain degree of conformational plasticity for the N-terminal b-sheet.…”

Proton-detected scalar coupling based assignment strategies in MAS solid-state NMR spectroscopy applied to perdeuterated proteins

“…Crystallization was performed by pH-shift using a 5 mg/ml protein solution in a crystallization buffer containing H 2 O:D 2 O at a ratio of 1:9. Paramagnetic relaxation enhancement (PRE) was used in order to decrease the recycle delay of the experiment [40,41]. For that purpose, [Cu(edta)] 2À was added upon crystallization at a concentration of 75 and 150 mM as described earlier [41].…”

“…Paramagnetic relaxation enhancement (PRE) was used in order to decrease the recycle delay of the experiment [40,41]. For that purpose, [Cu(edta)] 2À was added upon crystallization at a concentration of 75 and 150 mM as described earlier [41]. Crystals were packed by centrifugation into 3.2 and 4 mm rotors, respectively.…”

NMR Spectroscopy of Soluble Protein Complexes at One Mega‐Dalton and Beyond

“…[9,10] A stock solution of 400 mM Cu II -EDTA (Sigma Aldrich) in phosphate buffer containing 20% H 2 O, 80% D 2 O and 30% perdeuterated glycerol (Sigma Aldrich) was added to the proteasome samples to a final concentration of the paramagnetic agent of 60 mM. In order to optimize the recycle delay, 1 H longitudinal relaxation times T 1 were determined for the bulk amide region.…”