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

Abstract

High-sensitivity proton-detected experiments in solid-state NMR have been recently demonstrated in proton-diluted proteins as well as fully protonated samples under fast magic-angle spinning. One key element for performing successful proton detection is effective solvent suppression achieved by pulsed field gradients (PFG) and/or saturation pulses. Here we report a high-performance solvent suppression method that attenuates multiple solvent signals simultaneously by more than a factor of 10,000, achieved by an optimized combination of homospoil gradients and supercycled saturation pulses. This method, which we call Multiple Intense Solvent Suppression Intended for Sensitive Spectroscopic Investigation of Protonated Proteins, Instantly (MISSISSIPPI), can be applied without a PFG probe. It opens up new opportunities for two-dimensional heteronuclear correlation spectroscopy of hydrated proteins at natural abundance as well as high-sensitivity and multidimensional experimental investigation of protein-solvent interactions.

1H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

“…1 H‐detected 2D NH and CH spectra of iFD‐labeled [ 13 C, 15 N]ubiquitin in aqueous (100 % H 2 O) buffers, acquired at 60 kHz MAS and 950 MHz 1 H frequency using MISSISSIPI water suppression7 and PISSARRO decoupling,8 are shown in Figures 1 A, B. These spectra are very well resolved and feature an average linewidth of 0.1–0.2 ppm for H N and Hα at 950 MHz, which is excellent considering the high 1 H density of 80 % in iFD proteins, and constitutes a substantial average linewidth improvement of about 30–35 % compared to fully protonated ubiquitin (see Figure 1 C and the Supporting Information, Table S1; see also Section S2 for a quantitative 1 H population analysis and a discussion of the 1 H linewidth improvement).…”

NMR Spectroscopy of Soluble Protein Complexes at One Mega‐Dalton and Beyond

“…Due to the high content of water in FROSTY samples, suppression of the water resonance was achieved by implementing a pulse train of 4×15 ms with a rf field strength of 4-10 kHz and alternating phase during longitudinal 15 N magnetization. [11,12] The 2D 1 H-15 N (and 3D hCXhNH experiments adapted from [13,14] ) were designed in a constant-time fashion here to ensure reproducible dephasing of the water resonance as well as to yield optimal signal-to-noise ratios. Transmitters were placed at 4.7 ppm ( 1 H) and 119 ppm ( 15 N), respectively.…”

Festkörper‐NMR‐Spektroskopie mit Protonendetektion an fibrillären Proteinen und Membranproteinen

“…[1] Magnetization transfer was achieved via ramp-CP. For OmpG and Aß , CP yields better sensitivity in comparison to experiments in which INEPT steps are employed for magnetization transfer.…”

“…Long purge pulses were again employed for water suppression. [1] On-and off-resonance 13 C pulses were applied as soft rectangular and G3 shaped pulses, respectively. [4,5] Supporting Figure 1.…”

“…Proton detected NMR experiments. A) 2D H/N-correlation based on the MISSISSIPPI pulse scheme proposed by Zhou et al [1] B) HNCA. CP is employed for magnetization transfer between 1 H and 15 N. Water suppression was again achieved using long purge pulses.…”