jz7b02709_si_001.pdf (2.03 MB)
Rapid Quantitative Measurements of Paramagnetic Relaxation Enhancements in Cu(II)-Tagged Proteins by Proton-Detected Solid-State NMR Spectroscopy
journal contribution
posted on 2017-11-17, 00:00 authored by Dwaipayan Mukhopadhyay, Philippe S. Nadaud, Matthew D. Shannon, Christopher P. JaroniecWe
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.
History
Usage metrics
Categories
Keywords
PREproton-detected 2 D3 D correlation spectra13 C28CGB2 Hexperiment timeProton-Detected Solid-State NMR Spectroscopynatively diamagnetic proteinsbinding tagsParamagnetic Relaxation EnhancementsH 2 Oelectron relaxation time1 HCubackbone amide 15 NRapid Quantitative Measurementsrelaxation enhancementsstructure determination15 N-labeled protein back-exchanged
Licence
Exports
RefWorks
BibTeX
Ref. manager
Endnote
DataCite
NLM
DC