The benefit of combining in-cell solid-state dynamic nuclear polarization (DNP) NMR and cryogenic temperatures is providing sufficient signal/noise and preservation of bacterial integrity via cryoprotection to enable in situ biophysical studies of antimicrobial peptides. The radical source required for DNP was delivered into cells by adding a nitroxide-tagged peptide based on the antimicrobial peptide maculatin 1.1 (Mac1). In this study, the structure, localization, and signal enhancement properties of a single (T-MacW) and double (T-T-MacW) TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin-labeled Mac1 analogs were determined within micelles or lipid vesicles. The solution NMR and circular dichroism results showed that the spin-labeled peptides adopted helical structures in contact with micelles. The peptides behaved as an isolated radical source in the presence of multilamellar vesicles, and the electron paramagnetic resonance (EPR) electron-electron distance for the doubly spin-labeled peptide was ∼1 nm. The strongest paramagnetic relaxation enhancement (PRE) was observed for the lipid NMR signals near the glycerol-carbonyl backbone and was stronger for the doubly spin-labeled peptide. Molecular dynamics simulation of the T-T-MacW radical source in phospholipid bilayers supported the EPR and PRE observations while providing further structural insights. Overall, the T-T-MacW peptide achieved better ¹³C and ¹⁵N signal NMR enhancements and ¹H spin-lattice T₁ relaxation than T-MacW.

将细胞内固态动态核极化 (DNP) NMR 和低温相结合的好处是通过低温保护提供足够的信号/噪声和保持细菌完整性，从而能够对抗菌肽进行原位生物物理研究。通过添加基于抗菌肽 maculatin 1.1 (Mac1) 的氮氧化物标记肽，将 DNP 所需的自由基源递送到细胞中。在本研究中，单 (T-MacW) 和双 (TT-MacW) TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-) 的结构、定位和信号增强特性羧酸）自旋标记的 Mac1 类似物在胶束或脂质囊泡中测定。溶液核磁共振和圆二色性结果表明，自旋标记的肽与胶束接触时采用螺旋结构。在多层囊泡存在下，肽表现为孤立的自由基源，双自旋标记肽的电子顺磁共振 (EPR) 电子-电子距离约为 1 nm。对于甘油-羰基骨架附近的脂质核磁共振信号，观察到最强的顺磁弛豫增强 (PRE)，对于双自旋标记的肽则更强。磷脂双层中 TT-MacW 自由基源的分子动力学模拟支持 EPR 和 PRE 观察，同时提供进一步的结构见解。总体而言，TT-MacW 肽取得了更好的效果 对于甘油-羰基骨架附近的脂质核磁共振信号，观察到最强的顺磁弛豫增强 (PRE)，对于双自旋标记的肽则更强。磷脂双层中 TT-MacW 自由基源的分子动力学模拟支持 EPR 和 PRE 观察，同时提供进一步的结构见解。总体而言，TT-MacW 肽取得了更好的效果 对于甘油-羰基骨架附近的脂质核磁共振信号，观察到最强的顺磁弛豫增强 (PRE)，对于双自旋标记的肽则更强。磷脂双层中 TT-MacW 自由基源的分子动力学模拟支持 EPR 和 PRE 观察，同时提供进一步的结构见解。总体而言，TT-MacW 肽取得了更好的效果¹³ C 和¹⁵ N 信号 NMR 增强和¹ H 自旋晶格 T ₁弛豫比 T-MacW。