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

Abstract

Sensitivity and resolution together determine the quality of NMR spectra in biological solids. For high-resolution structure determination with solid-state NMR, protondetection 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 1GHz spectrometer for methyl resonances. Variable degrees of proton densities are tested on microcrystalline samples of the α-spectrin SH3 domain with selectively protonated methyl isotopomers (CH 3 , CH 2 D, CHD 2) 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.

Impact of Magnetic Field Strength on Resolution and Sensitivity of Proton Resonances in Biological Solids

“…Again, higher magnetic fields facilitate MAS-induced averaging of proton dipolar couplings. (11,12) Figure 5. (A) Simulated intensities for methyl protons of L34δ2, V44γ1, and V53γ1 calculated by assuming B0 fields of 500 MHz and 1 GHz and assuming that only valine and leucine methyl groups are labeled as 13 CH3 (while the rest of the protein is deuterated) for the αSH3 domain sample.…”

“…Above an MAS frequency of 70 kHz, 13 CH3 isotopomers (4,15−17) yield the best sensitivity depending on the density of the proton spin system. (12) The maximum achievable rotation frequency of an MAS rotor is limited by the speed of sound on the rotor surface. (18) Higher rotation frequencies can therefore only be obtained for ever smaller diameter rotors.…”

Proton Detected Solid-State NMR of Membrane Proteins at 28 Tesla (1.2 GHz) and 100 kHz Magic-Angle Spinning

“…A complementary strategy to proton dilution is to increase the MAS frequency [ 14 , 15 , 16 ] and the magnetic field in order to reduce spin relaxation occurring via spin flip-flops [ 17 , 18 ]. This places less stringent demands on proton dilution [ 19 , 20 , 21 , 22 ] and results in high sensitivity despite a reduction in sample amount to only 2 to 3 mg for 1.3 mm rotors spun up to 60 kHz MAS, as previously reviewed [ 23 ].…”

Towards a native environment: structure and function of membrane proteins in lipid bilayers by NMR

“… 80 The optimal methyl protonation level at different MAS frequencies was investigated by Reif's group. 81 With the use of selective – 13 CHD 2 methyl labelling, Griffin and coworkers extracted 1 H– 1 H distances in the conductance domain of influenza A M2. 33 Fig.…”

“…16, 28, 110-112 113 . Meier, Reif, and other groups are showing accumulating evidence that the quality of proton detected spectra continues to benefit from the increase in available spinning frequency 18,19,81,[114][115][116][117] . For example, the suppression of homogeneous proton-proton dipolar coupling will still benefit from increasing the MAS rate even beyond 300 kHz 118 , meaning further hardware development is still important and urgent 119 .…”