Solid-state NMR sequential assignments of the N-terminal domain of HpDnaB helicase

Abstract

We present solid-state NMR assignments of the N-terminal domain of the DnaB helicase from Helicobacter pylori (153 residues) in its microcrystalline form. We use a sequential resonance assignment strategy based on three-dimensional NMR experiments. The resonance assignments obtained are compared with automated resonance assignments computed with the ssFLYA algorithm. An analysis of the (13)C secondary chemical shifts determines the position of the secondary structure elements in this α-helical protein.

Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid‐State NMR Spectroscopy

“…15 N, 13 CNCA spectra are provided in Figure S3. Ap artial sequential resonance assignment of HpDnaB was obtained on the basis of the assignment of the N-terminal domain (NTD) [22] and from 3D experiments,a sd escribed in the Supporting Information ( Figure S4). Ap artial sequential resonance assignment of HpDnaB was obtained on the basis of the assignment of the N-terminal domain (NTD) [22] and from 3D experiments,a sd escribed in the Supporting Information ( Figure S4).…”

“…The 13 Cl inewidth is approximately 0.5 ppm for free as well as for AMP-PNP bound protein and indicates aw ell-ordered molecular conformation. [22,23] 25 %o ft he backbone atoms are assigned as detailed in Table S1. [22,23] 25 %o ft he backbone atoms are assigned as detailed in Table S1.…”

Beobachtung von ssDNA‐Bindung an die DnaB‐Helikase von Helicobacter pylori mittels Festkörper‐NMR‐Spektroskopie

“…0.5 ppm für freies sowie für AMP‐PNP‐gebundenes Protein, was auf eine wohlgeordnete molekulare Konformation hindeutet. Eine teilweise, sequentielle Resonanzzuordnung von Hp DnaB wurde auf Basis der Zuordnung der N‐terminalen Domäne (NTD) und durch 3D‐Experimente erzielt, wie in den Hintergrundinformationen beschrieben (Abbildung S4) . 25 % der Atome des Rückgrates konnten zugeordnet werden, vgl.…”

On the use of ultracentrifugal devices for routine sample preparation in biomolecular magic-angle-spinning NMR

“…It is designed to balance the need to maximize the signal/noise, maintain biological relevance and achieve easy reproducibility. We note that such devices are successfully used by ourselves (Van der Wel et al 2007; Li et al 2011; Hoop et al 2012; Hoop et al 2014; Mandal et al 2015; Mandal and Van der Wel 2016) and various other groups (Böckmann et al 2009; Gardiennet et al 2012; Bertini et al 2013; Kunert et al 2014; Wiegand et al 2015; Wiegand et al 2016b), but that there are many others that have not adopted this approach.…”