Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR – practical aspects

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dc.contributor.authorBell, Dallas
dc.contributor.authorLindemann, Florian
dc.contributor.authorGerland, Lisa
dc.contributor.authorAucharova, Hanna
dc.contributor.authorKlein, Alexander
dc.contributor.authorFriedrich, Daniel
dc.contributor.authorHiller, Matthias
dc.contributor.authorGrohe, Kristof
dc.contributor.authorMeier, Tobias
dc.contributor.authorvan Rossum, Barth
dc.contributor.authorDiehl, Anne
dc.contributor.authorHughes, Jon
dc.contributor.authorMueller, Leonard J.
dc.contributor.authorLinser, Rasmus
dc.contributor.authorMiller, Anne-Frances
dc.contributor.authorOschkinat, Hartmut
dc.date.accessioned2026-01-15T07:58:34Z
dc.date.available2026-01-15T07:58:34Z
dc.date.issued2024-06-21
dc.description.abstractSolution 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.en
dc.identifier.urihttp://hdl.handle.net/2003/44678
dc.language.isoen
dc.relation.ispartofseriesJournal of biomolecular NMR; 78(3)
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectProtein NMRen
dc.subjectDense phaseen
dc.subjectUltracentrifugationen
dc.subjectMagic-angle-spinningen
dc.subjectSolid-state NMRen
dc.subject.ddc540
dc.subject.rswkProteine
dc.subject.rswkFestkörper-NMR-Spektroskopie
dc.subject.rswkUltrazentrifugieren
dc.subject.rswkMAS-NMR-Spektroskopie
dc.titleSedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR – practical aspectsen
dc.typeText
dc.type.publicationtypeArticle
dcterms.accessRightsopen access
eldorado.dnb.deposittrue
eldorado.doi.registerfalse
eldorado.openaire.projectidentifierinfo:eu-repo/grantAgreement/EC/HE/101082494/EU/Fast-MAS Solid-State NMR as a Bypass to High-Molecular-Weight Proteins in Solution/bypassNMR/
eldorado.secondarypublicationtrue
eldorado.secondarypublication.primarycitationBell, D., Lindemann, F., Gerland, L. et al. Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR – practical aspects. J Biomol NMR 78, 179–192 (2024). https://doi.org/10.1007/s10858-024-00444-9
eldorado.secondarypublication.primaryidentifierhttps://doi.org/10.1007/s10858-024-00444-9

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