Department of Physics and Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada.
Department of Chemistry, North Carolina State University, Raleigh, NC, USA.
Solid State Nucl Magn Reson. 2019 Aug;100:92-101. doi: 10.1016/j.ssnmr.2019.04.003. Epub 2019 Apr 17.
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.
一种新型的动态核极化（DNP）NMR 极化剂 ToSMTSL-PTE 已被合成、表征，并用于增强脂质重组整合膜蛋白视紫红质（PR）的固态 NMR 信号。与传统的 TOTAPOL 分散在玻璃态甘油/水基质中的制剂相比，无基质 PR 制剂用于 DNP 可将 NMR 信号的绝对灵敏度提高约 4 倍。在 400 MHz/263 GHz 和 600 MHz/395 GHz 下测量的 DNP 增强显示出强烈的场依赖性，但在两个场中仍然适中，与用 ToSMTSL 共价修饰的 PR 获得的增强相当。在溶液和脂质双层中用 ToSMTSL-PTE 进行的连续波（CW）X 波段电子顺磁共振（EPR）实验表明，连接单氮氧自由基的连接体的不利构象变化可能是中等 DNP 增强的原因之一。此外，差示扫描量热法（DSC）和 CW EPR 实验表明，当极化剂浓度增加到 20 mol%以最大化 DNP 增强时，ToSMTSL-PTE 在 DMPC:DMPA 双层中的分布不均匀和/或部分聚集的可能性。因此，脂质双层中基于脂质的双自由基的构象变化和不均匀分布成为进一步开发这种无基质方法用于膜蛋白 DNP 时需要考虑的重要因素。
