A novel Dynamic Nuclear Polarization (DNP) NMR polarizing agent ToSMTSL-PTE representing a phospholipid with a biradical TOTAPOL tethered to the polar head group has been synthesized, characterized, and employed to enhance solid-state Nuclear Magnetic Resonance (SSNMR) signal of a lipid-reconstituted integral membrane protein proteorhodopsin (PR). A matrix-free PR formulation for DNP improved the absolute sensitivity of NMR signal by a factor of ca. 4 compared to a conventional preparation with TOTAPOL dispersed in a glassy glycerol/water matrix. DNP enhancements measured at 400 MHz/263 GHz and 600 MHz/395 GHz showed a strong field dependence but remained moderate at both fields, and comparable to those obtained for PR covalently modified with ToSMTSL. Additional continuous wave (CW) X-band electron paramagnetic resonance (EPR) experiments with ToSMTSL-PTE in solutions and in lipid bilayers revealed that an unfavorable conformational change of the linker connecting mononitroxides could be one of the reasons for moderate DNP enhancements. Further, differential scanning calorimetry (DSC) and CW EPR experiments indicated an inhomogeneous distribution and/or a possibility of a partial aggregation of ToSMTSL-PTE in DMPC:DMPA bilayers when the concentration of the polarizing agent was increased to 20 mol% to maximize the DNP enhancement. Thus, conformational changes and an inhomogeneous distribution of the lipid-based biradicals in lipid bilayers emerged as important factors to consider for further development of this matrix-free approach for DNP of membrane proteins.

合成，表征并用于增强脂质的固态核磁共振（SSNMR）信号的新型动态核极化（DNP）NMR极化剂ToSMTSL-PTE代表具有双极性TOTAPOL拴在极性头基上的磷脂重组膜蛋白蛋白视紫红质（PR）。用于DNP的无基质PR配方将NMR信号的绝对灵敏度提高了约两倍。与将TOTAPOL分散在玻璃状甘油/水基质中的常规制备方法相比，图4所示的制备方法与图4的制备方法不同。在400 MHz / 263 GHz和600 MHz / 395 GHz处测得的DNP增强表现出很强的场依赖性，但在两个场上均保持中等水平，与通过ToSMTSL共价修饰的PR获得的DNP增强相当。使用ToSMTSL-PTE在溶液和脂质双层中进行的其他连续波（CW）X波段电子顺磁共振（EPR）实验表明，连接一氧化氮的接头构象发生不利变化可能是DNP适度增强的原因之一。此外，差示扫描量热法（DSC）和CW EPR实验表明DMPC中ToSMTSL-PTE的分布不均匀和/或部分聚集的可能性：当偏振剂的浓度增加到20 mol％以最大化DNP增强时，DMPA双层。因此，脂质双层中基于脂质的双基团的构象变化和不均匀分布已成为考虑进一步开发这种无基质方法用于膜蛋白DNP的重要因素。