Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS solid-state NMR spectroscopy

Abstract

We present a systematic study of the effect of the level of exchangeable protons on the observed amide proton linewidth obtained in perdeuterated proteins. Decreasing the amount of D 2 O employed in the crystallization buffer from 90 to 0%, we observe a fourfold increase in linewidth for both 1 H and 15 N resonances. At the same time, we find a gradual increase in the signal-to-noise ratio (SNR) for 1 H-15 N correlations in dipolar coupling based experiments for H 2 O concentrations of up to 40%. Beyond 40%, a significant reduction in SNR is observed. Scalarcoupling based 1 H-15 N correlation experiments yield a nearly constant SNR for samples prepared with B30% H 2 O. Samples in which more H 2 O is employed for crystallization show a significantly reduced NMR intensity. Calculation of the SNR by taking into account the reduction in 1 H T 1 in samples containing more protons (SNR per unit time), yields a maximum SNR for samples crystallized using 30 and 40% H 2 O for scalar and dipolar coupling based experiments, respectively. A sensitivity gain of 3.8 is obtained by increasing the H 2 O concentration from 10 to 40% in the CP based experiment, whereas the linewidth only becomes 1.5 times broader. In general, we find that CP is more favorable compared to INEPT based transfer when the number of possible 1 H, 1 H interactions increases. At low levels of deuteration (C60% H 2 O in the crystallization buffer), resonances from rigid residues are broadened beyond detection. All experiments are carried out at MAS frequency of 24 kHz employing perdeuterated samples of the chicken a-spectrin SH3 domain.

Proton‐Detected Solid‐State NMR Spectroscopy of Fibrillar and Membrane Proteins

“…This is in agreement to what has been found previously for a microcrystalline preparation of the α-spectrin SH3 domain. [11] Supporting Figure 2. A and B) Comparison of H/N correlation spectra of Aß 1-40 fibrils prepared without and with of 75 mM Cu(edta), respectively.…”

Narrow carbonyl resonances in proton-diluted proteins facilitate NMR assignments in the solid-state

“…Introducing a second chemical shift dimension facilitates resolving chemical shift degeneracies. Extensive deuteration is achieved by crystallizing the perdeuterated protein in a buffer which contains H 2 O and D 2 O in a ratio of around 10-30% (Akbey et al 2009;Chevelkov et al 2006). …”

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

“…Despite early proof-of-principle papers (5), this approach only became popular with the realization that amide sites must be only partially reprotonated (typically 10-30% back exchange) (6)(7)(8) to yield well-resolved 1 H spectra. This represented a significant compromise in sensitivity to gain resolution, and effectively made the determination of internuclear distances impractical, with few exceptions (9)(10)(11).…”