期刊
JOURNAL OF MAGNETIC RESONANCE
卷 332, 期 -, 页码 -出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jmr.2021.107075
关键词
Solid-state NMR; Spin labeling; Paramagnetic Relaxation Enhancement; Molecular modeling; Motor protein
资金
- Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) [455240421]
- Deutsche Forschungsgemeinschaft (DFG, Heisenberg fellowship) [455238107]
- DFG postdoctoral fellowship [KU 3510/1-1]
- ETH Research Grant [ETH-43 17-2]
Labeling biomolecules with a paramagnetic probe for NMR spectroscopy allows determining long-range distance restraints, aiding in the structure determination of large proteins and protein complexes. The success of site-directed labeling of bacterial DnaB helicase with maleimide tags was confirmed through EPR experiments, leading to the extraction of site-specific PREs for further characterization. Comparison of experimental PREs and computational modeling of nitroxide and Gd3+ tags revealed differences in the blind sphere size around the paramagnetic center.
Labeling of biomolecules with a paramagnetic probe for nuclear magnetic resonance (NMR) spectroscopy enables determining long-range distance restraints, which are otherwise not accessible by classically used dipolar coupling-based NMR approaches. Distance restraints derived from paramagnetic relaxation enhancements (PREs) can facilitate the structure determination of large proteins and protein complexes. We herein present the site-directed labeling of the large oligomeric bacterial DnaB helicase from Helicobacter pylori with cysteine-reactive maleimide tags carrying either a nitroxide radical or a lanthanide ion. The success of the labeling reaction was followed by quantitative continuous-wave electron paramagnetic resonance (EPR) experiments performed on the nitroxide-labeled protein. PREs were extracted site-specifically from 2D and 3D solid-state NMR spectra. A good agreement with predicted PRE values, derived by computational modeling of nitroxide and Gd3+ tags in the low-resolution DnaB crystal structure, was found. Comparison of experimental PREs and model-predicted spin label-nucleus distances indicated that the size of the blind sphere around the paramagnetic center, in which NMR resonances are not detected, is slightly larger for Gd3+ (similar to 14 angstrom) than for nitroxide (similar to 11 angstrom) in C-13-detected 2D spectra of DnaB. We also present Gd3+-Gd3+ dipolar electron-electron resonance EPR experiments on DnaB supporting the conclusion that DnaB was present as a hexameric assembly. (C) 2021 The Author(s). Published by Elsevier Inc.
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