Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR
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Date
2021-11
Publication Type
Journal Article
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yes
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Abstract
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⁺ (∼14 Å) than for nitroxide (∼11 Å) in ¹³C-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.
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published
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Journal / series
Volume
332
Pages / Article No.
107075
Publisher
Elsevier
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Edition / version
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Date collected
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Subject
Solid-state NMR; Spin labeling; Paramagnetic Relaxation Enhancement; Molecular modeling; Motor protein
Organisational unit
02515 - Laboratorium für Physikalische Chemie / Laboratory of Physical Chemistry