Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location.

摘要: Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation sample preparation protocols for optimizing the sensitivity spectra membrane-bound peptides proteins at cryogenic temperatures ~110 K. We show that mixing radical with membrane by direct titration instead centrifugation gives significant boost enhancement. quantify relative between AMUPol TOTAPOL, two commonly used radicals, deuterated protonated lipid membranes. shows ~fourfold higher than while does not give net membrane. Overall, ~100 fold microwave-on microwave-off can be achieved on lipid-rich membranes containing conformationally disordered peptides, absolute gains 105-160 obtained low-temperature high-temperature non-DNP spectra. also measured relaxation signals TOTAPOL AMUPol, determine depths these in bilayer. Our data indicate bimodal distribution both surface-bound fraction where nitroxides lie ~10 A from surface. appears membrane-embedded AMUPol. These results should useful under conditions provide insights into how biradicals interact phospholipid