Field and magic angle spinning frequency dependence of proton resonances in rotating solids.

Proton detection in solid state NMR is continuously developing and allows one to gain new insights in structural biology. Overall, this progress is a result of the synergy between hardware development, new NMR methodology and new isotope labeling strategies, to name a few factors. Even though current developments are rapid, it is worthwhile to summarize what can currently be achieved employing proton detection in biological solids. We illustrate this by analysing the signal-to-noise ratio (SNR) for spectra obtained for a microcrystalline α-spectrin SH3 domain protein sample by (i) employing different degrees of chemical dilution to replace protons by incorporating deuterons in different sites, by (ii) variation of the magic angle spinning (MAS) frequencies between 20 and 110 kHz, and by (iii) variation of the static magnetic field B0. The experimental SNR values are validated with numerical simulations employing up to 9 proton spins. Although in reality a protein would contain far more than 9 protons, in a deuterated environment this is a sufficient number to achieve satisfactory simulations consistent with the experimental data. The key results of this analysis are (i) with current hardware, deuteration is still necessary to record spectra of optimum quality; (ii) 13CH3 isotopomers for methyl groups yield the best SNR when MAS frequencies above 100 kHz are available; and (iii) sensitivity increases with a factor beyond B0 3/2 with the static magnetic field due to a transition of proton-proton dipolar interactions from a strong to a weak coupling limit

Magic-angle spinning frequencies beyond 300 kHz are necessary to yield maximum sensitivity in selectively methyl protonated protein samples in solid-state NMR.

In the last decade, proton detection in magic-angle spinning (MAS) solid-state NMR became a popular strategy for biomolecular structure determination. In particular, probe technology has experienced tremendous progress with smaller and smaller diameter rotors achieving ever higher MAS frequencies. MAS rotation frequencies beyond 100 kHz allow to observe and assign protons in fully protonated samples. In these experiments, resolution is however compromised as homogeneous proton-proton dipolar coupling interactions are not completely averaged out. Using a combination of experiments and simulations, we analyze the MAS frequency-dependent intensities of the 1H,13C methyl correlation peaks of a selectively methyl protonated (CH3) microcrystalline sample of the chicken α-spectrin SH3 domain (α-SH3). Extensive simulations involving nine spins employing the program SIMPSON allow to predict the MAS frequency dependence of the proton intensities. The experimental results are used to validate the simulations. As quantitative measure, we determine the characteristic MAS frequency, which is necessary to obtain >50% of the maximum achievable sensitivity. Our results show that this frequency is site-specific and strongly depends on the local methyl density. We find that the characteristic MAS frequency ranges from as low as 20 kHz up to 324 kHz with the average value of 135 ± 88 kHz for this particular sample at a magnetic field strength of 11.7 T. Inclusion of side chain dynamics in the analysis reduces the average characteristic MAS frequency to 104 ± 68 kHz within the range of 11-261 kHz. In case, >80% of the maximum sensitivity shall be achieved, MAS rotation frequencies of 498 ± 370 and 310 ± 227 kHz are required with and without including side chains dynamics in the analysis, respectively

MAS dependent sensitivity of different isotopomers in selectively methyl protonated protein samples in solid state NMR.

Sensitivity and resolution together determine the quality of NMR spectra in biological solids. For high-resolution structure determination with solid-state NMR, proton-detection emerged as an attractive strategy in the last few years. Recent progress in probe technology has extended the range of available MAS frequencies up to above 100 kHz, enabling the detection of resolved resonances from sidechain protons, which are important reporters of structure. Here we characterise the interplay between MAS frequency in the newly available range of 70-110 kHz and proton content on the spectral quality obtainable on a 1 GHz spectrometer for methyl resonances. Variable degrees of proton densities are tested on microcrystalline samples of the alpha-spectrin SH3 domain with selectively protonated methyl isotopomers (CH3, CH2D, CHD2) in a perdeuterated matrix. The experimental results are supported by simulations that allow the prediction of the sensitivity outside this experimental frequency window. Our results facilitate the selection of the appropriate labelling scheme at a given MAS rotation frequency

Limits of resolution and sensitivity of proton detected MAS solid-state NMR experiments at 111 kHz in deuterated and protonated proteins.

MAS solid-state NMR is capable of determining structures of protonated solid proteins using proton-detected experiments. These experiments are performed at MAS rotation frequency of around 110 kHz, employing 0.5 mg of material. Here, we compare (1)H, (13)C correlation spectra obtained from protonated and deuterated microcrystalline proteins at MAS rotation frequency of 111 kHz, and show that the spectral quality obtained from deuterated samples is superior to those acquired using protonated samples in terms of resolution and sensitivity. In comparison to protonated samples, spectra obtained from deuterated samples yield a gain in resolution on the order of 3 and 2 in the proton and carbon dimensions, respectively. Additionally, the spectrum from the deuterated sample yields approximately 2-3 times more sensitivity compared to the spectrum of a protonated sample. This gain could be further increased by a factor of 2 by making use of stereospecific precursors for biosynthesis. Although the overall resolution and sensitivity of (1)H, (13)C correlation spectra obtained using protonated solid samples with rotation frequencies on the order of 110 kHz is high, the spectral quality is still poor when compared to the deuterated samples. We believe that experiments involving large protein complexes in which sensitivity is limiting will benefit from the application of deuteration schemes

NMR Investigation of Chloromethane Complexes of Cryptophane-A and Its Analogue with Butoxy Groups

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