Sensitive High Resolution Inverse Detection NMR Spectroscopy of Proteins in the Solid State

Abstract

A new indirect detection scheme for obtaining (15)N/(1)H shift correlation spectra in crystalline proteins is described. Excellent water suppression is achieved without the need for pulsed field gradients, and using only a 2-step phase cycle. Careful attention to overall NMR instrument stability was found critical for obtaining the best resolution and sensitivity. Magnetic dilution by deuteration of the protein in combination with high-speed magic angle spinning produces (1)H resonances averaging only 0.22 ppm in width, and in some cases lines as narrow as 0.17 ppm are obtained. In application to two different polymorphs of ubiquitin, structure dependent differences in both (15)N and (1)H amide chemical shifts are observed. In one case, distinct shifts for different molecules in the asymmetric unit are seen, and all differ substantially from solution NMR shifts. A gain of 7 in sensitivity makes the method competitive with solution NMR as long as nanocrystalline samples are available.

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

“…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].…”

“…Therefore the water resonance is more than 10,000 times stronger than a 13 C or 15 N-edited protein proton resonance; thus water and other solvent signals must be suppressed by four orders of magnitude to utilize the full receiver dynamic range for the protein signals. Previously reported solvent suppression schemes yield suppression by two to three orders of magnitude for a single resonance line (such as water), achieved by employing either pulsed field gradients (PFGs) [9] or saturation pulses [10]. We had extended the method of Paulson and Zilm [10] to achieve simultaneous suppression of multiple solvent signals, by using a long, moderate-power saturation pulse [13].…”

Crystal Polymorphism of Protein GB1 Examined by Solid-State NMR Spectroscopy and X-ray Diffraction

“…[14][15][16][17][18] One such study by Paulson et al showed that nanocrystalline sample preparations of ubiquitin yielded three different sets of 1 H- 15 N correlation spectra, resulting from three different polymorphs. 10 Subsequently Siedel et al assigned the C', CA and CB chemical shifts for two of these polymorphs. 11 Both of these studies focused on chemical shift differences among the backbone resonances.…”

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.…”