Fabien Aussenac

Towards high-resolution 1H-NMR in biological membranes: magic angle spinning of bicelles

Proton line narrowing in biomembranes spun at the magic angle, for spinning speeds greater than 7 kHz, was investigated in two ways: increasing the field strength from 200 to 800 MHz and changing the membrane fluidity. The resolution that one can obtain on natural lipid membranes under the form of liposomes is 0.019 ppm at 800 MHz. On the other hand, spinning bicelles (disk-like model membranes made of synthetic long and short chain lipids) at the magic angle decreases the line width by an additional factor of 3 provided the bicelle is subjected to large orientational disorder. This leads to proton line widths of the order of 6 Hz at 500 MHz. The conjunction of high field, magic angle spinning and use of bicelle membranes should prove to be useful to solve membrane protein structure in a membrane environment.

Temperature dependence of cross-effect dynamic nuclear polarization in rotating solids: advantages of elevated temperatures

Dynamic nuclear polarization exploits electron spin polarization to boost signal-to-noise in magic-angle-spinning (MAS) NMR, creating new opportunities in materials science, structural biology, and metabolomics studies. Since protein NMR spectra recorded under DNP conditions can show improved spectral resolution at 180-200 K compared to 110 K, we investigate the effects of AMUPol and various deuterated TOTAPOL isotopologues on sensitivity and spectral resolution at these temperatures, using proline and reproducibly prepared SH3 domain samples. The TOTAPOL deuteration pattern is optimized for protein DNP MAS NMR, and signal-to-noise per unit time measurements demonstrate the high value of TOTAPOL isotopologues for Protein DNP MAS NMR at 180-200 K. The combined effects of enhancement, depolarization, and proton longitudinal relaxation are surprisingly sample-specific. At 200 K, DNP on SH3 domain standard samples yields a 15-fold increase in signal-to-noise over a sample without radicals. 2D and 3D NCACX/NCOCX spectra were recorded at 200 K within 1 and 13 hours, respectively. Decreasing enhancements with increasing 2H-content at the CH2 sites of the TEMPO rings in CD3-TOTAPOL highlight the importance of protons in a sphere of 4-6 Å around the nitroxyl group, presumably for polarization pickup from electron spins.