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Rapid Quantitative Measurements of Paramagnetic Relaxation Enhancements in Cu(II)-Tagged Proteins by Proton-Detected Solid-State NMR Spectroscopy
December 07 2017
November 20 2017
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Abstract
<p class="first" id="P1">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 Cu
<sup>2+</sup>-binding tags. Specifically, using protein GB1 K28C-EDTA-Cu
<sup>2+</sup> mutant as a model, we show the determination of backbone amide
<sup>15</sup>N longitudinal and
<sup>1</sup>H 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
<sup>2</sup>H,
<sup>13</sup>C,
<sup>15</sup>N-labeled protein back-exchanged in H
<sub>2</sub>O. Additionally, we show that the electron relaxation time for the Cu
<sup>2+</sup> 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.
</p><p id="P2">
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