Dynamic Nuclear Polarization/Solid‐State NMR Spectroscopy of Membrane Polypeptides: Free‐Radical Optimization for Matrix‐Free Lipid Bilayer Samples

Abstract

Dynamic nuclear polarization (DNP) boosts the sensitivity of NMR spectroscopy by orders of magnitude and makes investigations previously out of scope possible. For magic-angle-spinning (MAS) solid-state NMR spectroscopy studies, the samples are typically mixed with biradicals dissolved in a glass-forming solvent and are investigated at cryotemperatures. Herein, we present new biradical polarizing agents developed for matrix-free samples such as supported lipid bilayers, which are systems widely used for the investigation of membrane polypeptides of high biomedical importance. A series of 11 biradicals with different structures, geometries, and physicochemical properties were comprehensively tested for DNP performance in lipid bilayers, some of them developed specifically for DNP investigations of membranes. The membrane-anchored biradicals PyPol-C16, AMUPOL-cholesterol, and bTurea-C16 were found to exhibit improved g-tensor alignment, inter-radical distance, and dispersion. Consequently, these biradicals show the highest signal enhancement factors so far obtained for matrix-free membranes or other matrix-free samples and may potentially shorten NMR acquisition times by three orders of magnitude. Furthermore, the optimal biradical-to-lipid ratio, sample deuteration, and membrane lipid composition were determined under static and MAS conditions. To rationalize biradical performance better, DNP enhancement was measured by using the C and N signals of lipids and a peptide as a function of the biradical concentration, DNP build-up time, resonance line width, quenching effect, microwave power, and MAS frequency.

Continuous wave electron paramagnetic resonance of nitroxide biradicals in fluid solution

“…5 A variety of dinitroxides have been designed for use as polarizing agents in dynamic nuclear polarization (DNP). [6][7][8][9][10] Dinitroxides are used in DNP in preference to monoradicals due to the cross-effect (CE) process. 11 Dipolar interactions between the 2 nitroxides can be designed to match the nuclear Zeeman frequency.…”

Natural Isotopic Abundance 13C and 15N Multidimensional Solid-State NMR Enabled by Dynamic Nuclear Polarization

“…hydrophilicity) of the polarization agent or to target it to specific parts of a sample. [32][33][34][35][36][37][38][39][40] The polarization agent has to be homogeneously distributed in the sample to achieve efficient MAS-DNP. It is thus typically dissolved in a glass-forming mixture (containing glycerol or DMSO for instance) that can be used to suspend or impregnate the sample of interest.…”

Dynamic Nuclear Polarization / solid-state NMR of membranes. Thermal effects and sample geometry

“…DNP can overcome many of the concomitant sensitivity limitations and has indeed been used to study biomacromolecules [17,27]. However, the signal enhancements obtained when cells or membranes were investigated lag behind by about an order of magnitude to those obtained in isotropic environments and the reasons for this difference are only slowly understood [3,4,6,7,[28][29][30][31][32][33][34][35][36][37].…”

“…around 100 K) the sample is typically embedded in a glass forming matrix to ensure a homogenous distribution the paramagnetic polarizing agent [1,41]. For matrix-free samples such as stacks of membranes oriented on solid supports additional experimental considerations arise [37,42]. These samples are prepared in the absence of bulk water and have resulted in even lower DNP signal enhancements [26,37,43,44] .…”

“…For matrix-free samples such as stacks of membranes oriented on solid supports additional experimental considerations arise [37,42]. These samples are prepared in the absence of bulk water and have resulted in even lower DNP signal enhancements [26,37,43,44] . 1 2 3 4 5 6 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 For nitroxide-based polarizing agents used in such experiments the cross effect (CE) has been described as the most efficient polarization mechanism, which involves two coupled electron spins and a nearby proton were the difference in Larmor frequencies of the two electrons matches the Larmor frequency of the proton [1,45].…”