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 i

SUBMITTER: Mukhopadhyay D 

PROVIDER: S-EPMC5720925 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature