Spectral editing of intra- and inter-chain methyl–methyl NOEs in protein complexes

Abstract

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Backbone and methyl resonances assignment of the 87 kDa prefoldin from Pyrococcus horikoshii

“…The purification protocol of prefoldin subunits was adapted from (Okochi et al 2002) and is described in an earlier publication (Törner et al 2020). Subunits were expressed and purified separately, then combined and re-purified, allowing for combination of labelled/unlabelled subunits in one sample.…”

“…A 3D HCC HMQC-NOESY-HMQC experiment (Tugarinov et al 2005) was acquired at 70°C on a Bruker Avance III HD spectrometer equipped with a cryogenic probe and operating at a 1 H frequency of 950 MHz and methyl group assignment was performed by comparison with the crystal structure of PFD from Pyrococcus horikoshii (Ohtaki et al 2008) with the automated methyl assignment software MAGIC (Monneau et al 2017). These results were cross-validated or completed using a special labelling-scheme to distinguish inter-and intra-chain NOEs (Törner et al 2020). Furthermore 3D 'out and back' HCC, HC(C)C and HC(CC)C-experiments (Tugarinov et al 2003;Ayala et al 2009;Mas et al 2013) were acquired (at 70°C and a 1 H frequency of 950 MHz) in order to connect ( 13 C, 1 H)-methyl signals to backbone 13 Cα and 13 Cβ resonances using PFD-samples (~200 µM) labelled with linear…”

“…8 2a). Optimized 13 C-edited NOESY experiments combined with a specific labelling scheme on α and β subunit allowed to distinguish inter-from intra-chain NOEs in the crowded spectrum, thereby facilitating this step (Törner et al 2020). This distinction in β/β' assignment was transferred to the backbone via the connection of Cα /Cβ with methyl resonances (Fig.…”

Optimized precursor to simplify assignment transfer between backbone resonances and stereospecifically labelled valine and leucine methyl groups: application to human Hsp90 N-terminal domain

“…The 3D CCH HMQC-NOESY-HMQC NMR experiment (Tugarinov et al 2005;Törner et al 2020) was recorded over 3 days on a spectrometer operating at a 1 H frequency of 950 MHz using a 0.5 mM sample of U-[ 2 H, 15 N, 12…”

“…The (Kerfah et al 2015b, a;Törner et al 2020) , Ile-γ 2 (Ayala et al 2012) or Ala-β (Ayala et al 2009;Kerfah et al 2015a;Törner et al 2020), to backbone nuclei using a linear 13 C chain. In this study, we chose to label our sample on both leucine/valine pro-S and isoleucine-δ 1 methyl moieties, by adding Ile-δ 1 precursor (sodium (S)-2-hydroxy-2-(1',1'-…”