Solid‐State Protein‐Structure Determination with Proton‐Detected Triple‐Resonance 3D Magic‐Angle‐Spinning NMR Spectroscopy

Abstract

Magische Methoden: Die Kombination von schneller Drehung im magischen Winkel, Isotopenverdünnung und hoher Magnetfeldstärke ergibt gut aufgelöste Festkörper‐1H‐NMR‐Spektren, die effizient eingesetzt wurden, um Proteinstrukturen zu lösen. Mit den neuen Techniken genügen nun drei Tage für die Datensammlung, die Zuordnung der Signale zu den Protonen und die Erstellung einer hochaufgelösten Struktur von mikrokristallinem GB1.

High-performance solvent suppression for proton detected solid-state NMR

“…MISSISSIPPI-based solid-state 15 N-1 H heteronuclear correlation pulse sequence employing homospoil gradients and saturation pulses for efficient suppression of multiple solvent signals. Optionally, RFDR (the π pulse train and bracketing π/2 pulses on 1 H channel) or spin-diffusion (omitting the π pulses) can be used to study protein-solvent interactions as well as protonproton distance constraints within the protein [12,20]. For 2D acquisition, States-TPPI phase increment was applied to the π/2 pulse immediately following t 1 [23,24].…”

“…Natural abundance and 2 H, 13 C, 15 N-labeled β1 immunoglobulin binding domain of protein G (GB1) were prepared according to previously published procedures [12,14]. Nano-crystalline GB1 samples were precipitated with natural abundance 2-methyl-pentane-2,4-diol (MPD) and isopropanol (IPA) [14].…”

“…The strong proton dipolar coupling problem has been addressed with two approaches: (1) diluting the proton bath, (2) spinning fully protonated samples at ~40 kHz. The first approach yields very high resolution [8][9][10][11], and 1 H-1 H distance restraints as long as 9 Å have been detected, enabling the determination of a high-resolution protein structure [12]. The second approach is important for systems such as membrane proteins where deuteration and back-exchange may be challenging; it also permits all non-exchangeable protons to be investigated in a single experiment [13].…”

Proton-detected scalar coupling based assignment strategies in MAS solid-state NMR spectroscopy applied to perdeuterated proteins

“…Nevertheless, the overall acquisition times amounted to approximately 2 days (HNCO, sample A) and 2 days (HNCA, for sample B), and 4.5 days (HNCACB, sample B) due to the reduced proton abundance, resulting in an approximate signal-to-noise ratio of 45:1, 25:1, and 16:1, respectively. Recently, Rienstra and co-workers introduced a proton detected CON(H)H experiment in the context of determination of 1 H, 1 H distances [23]. The CON(H)H yields similar connectivity information as the HNCO, but relies on dipolar interactions to transfer magnetization.…”

“…In conventional solid-state NMR, 1 H detection is usually less favorable, as large dipolar couplings lead to substantial line broadening. We and others showed that this disadvantage can be circumvented by a reduction of the samples' proton density [16][17][18][19][20][21][22][23]. This allows to detect protons with high sensitivity and resolution.…”