Rapid Quantitative Measurements of Paramagnetic Relaxation Enhancements in Cu(II)-Tagged Proteins by Proton-Detected Solid-State NMR Spectroscopy

Abstract

We demonstrate rapid quantitative measurements of site-resolved paramagnetic relaxation enhancements (PREs), which are a source of valuable structural restraints corresponding to electron-nucleus distances in the ~10–20 Å regime, in solid-state nuclear magnetic resonance (NMR) spectra of proteins containing covalent Cu2+-binding tags. Specifically, using protein GB1 K28C-EDTA-Cu2+ mutant as a model, we show the determination of backbone amide 15N longitudinal and 1H transverse PREs within a few hours of experiment time based on proton-detected 2D or 3D correlation spectra recorded with magic-angle spinning frequencies ≥ ~60 kHz for samples containing ~10–50 nanomoles of 2H,13C,15N-labeled protein back-exchanged in H2O. Additionally, we show that the electron relaxation time for the Cu2+ center, needed to convert PREs into distances, can be estimated directly from the experimental data. Altogether, these results are important for establishing solid-state NMR based on paramagnetic-tagging as a routine tool for structure determination of natively diamagnetic proteins.

Accurate Electron–Nucleus Distances from Paramagnetic Relaxation Enhancements

“…In a previous study, R 1 ( 1 H) or R 2 ( 1 H) PREs generated by a Mn 2+ -EDTA tag were shown to give good quality distances in the range between 16 and 23 Å . That same study also reported good quality distances from the same EDTA tag loaded with Cu 2+ in the range between 9 and 18 Å. Cu 2+ tags have also successfully been used for distance measurements in the solid state in the range between 11 and 19 Å, with limited accuracy. − The data presented here allow more accurate distance measurements over a larger range than either of these transition metals. Furthermore, a high degree of accuracy is maintained for long distances, so that the useful range of distance measurements may extend beyond 23 Å in a larger protein complex.…”

Paramagnetic Chemical Probes for Studying Biological Macromolecules

“…647 R 1 PREs of 1 H spins are challenging to measure in solids due to spin diffusion effects, but R 2 PREs of 1 H can offer long-range distance restraints. 648 R 2 PREs by the SBM mechanism are largest for paramagnetic centers with slowly relaxing electrons such as the nitroxide spin label, which has also been demonstrated in GB1 by ligating cysteine residues with MTSL. 649 Measurement of PCSs generated by paramagnetic metal tags is more difficult in solids than in solution, because different orientations of the metal complex relative to the protein would lead to a range of different PCSs.…”

Structural Studies of Amyloid Fibrils by Paramagnetic Solid-State Nuclear Magnetic Resonance Spectroscopy

“…These measurements provide valuable information about amyloid structure, including data inaccessible by conventional SSNMR, and, remarkably, enable the determination of the protein fold in the huPrP23-144 amyloid core and protofilament assembly. This methodology is expected to be broadly applicable to other amyloid-forming proteins and can be further extended to other types of paramagnetic tags , and by incorporating rapid sample spinning and proton detection , for improved sensitivity.…”