Site-specific dynamic nuclear polarization in a Gd(iii)-labeled protein

Abstract

Dynamic nuclear polarization (DNP) of a biomolecule tagged with a polarizing agent has the potential to not only increase NMR sensitivity but also to provide spectroscopy specificity towards the tagging...

Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR

“…In comparison, the nitroxide tag leads to a slightly larger 13 C blind sphere in the 3D NCACB spectrum of 12 Å (Figure S5c and S8). These values compare favorably with the reported ones for the nitroxide in MTSL [61] or the Gd 3+ ion in dopants and metalloproteins [62,63], as well as in metal-ion DNP studies employing Gd 3+ -DOTA-M tags [26]. Noteworthy, the lengths of the linker connecting the protein backbone to the nitroxide (10.2 Å) or the Gd 3+ ion (12.5 Å) in the model structures is only slightly smaller than the size of the blind sphere determined in Figure 7.…”

“…The derivation of equation ( 1) is described in detail in Tamaki et al [18]. ICP is dependent on the paramagnetic relaxation rates of 1 H and 13 C in the rotating frame (Γ1ρ,H, Γ1ρ,C), IDARR is a function of the paramagnetic longitudinal relaxation rate of 13 The effective unpaired electron-nucleus distance, ̅ , was calculated based on the refined DnaB structure model and the simulated spin density distributions for Gd 3+ and the nitroxide radical according to [26]:…”

“…Additionally, diamagnetic proteins and nucleic acids [22] can be modified at specific positions with covalently bound paramagnetic tags [12,23], e.g. by using tags that react with the thiol groups of free cysteines which can be site-specifically introduced into proteins by mutagenesis [24][25][26][27]. In case of membrane proteins, transition metal ions can be attached to the phospholipid head groups of the membrane by chelating tags [28][29][30].…”

Targeted DNP for biomolecular solid-state NMR

“…Of course, many new developments are still necessary to move from the proof-of-concept stage to a robust and widely applicable solution. This includes developing DNP at higher magnetic fields, combining DNP-NMR data with computational approaches, 111 but also accessing sustainable helium spinning at ultra-low temperature (<100 K), and developing chemically persistent radicals for in-cell t DNP applications.…”

Trendbericht: Kernmagnetische Resonanz

“…33) Von Interesse dürfte die Entwicklung von 13 C-basierten Metabolomics mit hoher, Dissolution-DNP-gesteigerter Empfindlichkeit sein (siehe dazu Abschnitt Hyperpolarisation). 34) Weitere Entwicklungen mit paramagnetischen Gruppen sind etwa die ultraempfindliche aminosäurespezifische Detektion durch Gadolinium-Markierungen, 35) die 300-fache Empfindlichkeitssteigerung bei der Detektion der Iminoprotonen in RNA und DNA durch hyperpolarisiertes Wasser 36) sowie die Detektion von Lösungsmittelzugänglichkeit und damit vorübergehend existierender Strukturelemente in intrinsisch ungeordneten Proteinen (IDPs) durch Gadolinium-dotiertes Wasser. 37 41) Kürzlich ist es gelungen, die Struktur der transmembranen Domäne und einer Wirkstoffbindungsstelle des Sars-Cov-2-Virus-Hüllproteins E mit 2,1 Å Genauigkeit zu bestimmen.…”