Sedimentation of large, soluble proteins up to 140 kDa for
Dense phase
Magic-angle-spinning
Protein NMR
Solid-state NMR
Ultracentrifugation
Journal
Journal of biomolecular NMR
ISSN: 1573-5001
Titre abrégé: J Biomol NMR
Pays: Netherlands
ID NLM: 9110829
Informations de publication
Date de publication:
21 Jun 2024
21 Jun 2024
Historique:
received:
20
02
2024
accepted:
08
05
2024
medline:
21
6
2024
pubmed:
21
6
2024
entrez:
21
6
2024
Statut:
aheadofprint
Résumé
Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric β-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.
Identifiants
pubmed: 38904893
doi: 10.1007/s10858-024-00444-9
pii: 10.1007/s10858-024-00444-9
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Informations de copyright
© 2024. The Author(s).
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