Rapid Quantitative Measurements of Paramagnetic Relaxation
Enhancements in Cu(II)-Tagged Proteins by Proton-Detected Solid-State
NMR Spectroscopy
Dwaipayan Mukhopadhyay
Philippe S. Nadaud
Matthew D. Shannon
Christopher P. Jaroniec
10.1021/acs.jpclett.7b02709.s001
https://acs.figshare.com/articles/journal_contribution/Rapid_Quantitative_Measurements_of_Paramagnetic_Relaxation_Enhancements_in_Cu_II_-Tagged_Proteins_by_Proton-Detected_Solid-State_NMR_Spectroscopy/5616562
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.
2017-11-17 00:00:00
PRE
proton-detected 2 D
3 D correlation spectra
13 C
28C
GB
2 H
experiment time
Proton-Detected Solid-State NMR Spectroscopy
natively diamagnetic proteins
binding tags
Paramagnetic Relaxation Enhancements
H 2 O
electron relaxation time
1 H
Cu
backbone amide 15 N
Rapid Quantitative Measurements
relaxation enhancements
structure determination
15 N-labeled protein back-exchanged