Solid-State NMR Spectroscopy on Cellular Preparations Enhanced by Dynamic Nuclear Polarization
Solid-State NMR Spectroscopy on Cellular Preparations Enhanced by Dynamic Nuclear Polarization
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DOI：
10.1002/anie.201105984
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发表时间：
2012-01-01
影响因子：
16.6
通讯作者：
Baldus, Marc
中科院分区：
文献类型：
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作者：
Renault, Marie;Pawsey, Shane;Baldus, Marc
固体核磁共振谱为在原子水平上研究复杂的生物分子提供了越来越多的可能性。[1]膜相关蛋白是一个重要的靶点，膜相关蛋白在合成的双层分子中重组后可以用单核磁共振方法进行研究。虽然这样的准备可以检查感兴趣蛋白质的功能方面，但无法监测天然细胞环境对蛋白质结构和功能的影响。最近，我们引入了一种通用的方法，旨在通过魔角旋转(MAS)条件下的ss核磁共振来确定自然细胞环境中的复杂分子结构，包括完整的膜蛋白。[2，3]使用专门的样品制备路线，我们证明了可以在均匀的13C，15N标记的大肠杆菌全细胞(WC)和细胞膜(CE)的制备上获得高分辨率的ss核磁共振谱。在标准的单核磁共振实验条件下，CE和WC的形貌都保持不变，相应的13C和15N交叉极化(CP-MAS)谱随时间保持不变。然而，随着分子复杂性水平的提高，特别是在WC制备的情况下，光谱敏感性成为一个关键因素。近年来，动态核极化(DNP)已经发展成为提高多维单链核磁共振灵敏度的常规工具。[4]动态核极化在微/纳米晶体生物分子样品上获得了高达148倍的增强，包括淀粉样多肽和氚蛋白[5，6]，而对于膜包埋多肽、紫膜制剂和噬菌体，已有报道增强了18到46倍。[7，8]在这里，我们研究了使用DNP对13C，15N标记的大肠杆菌WC生产完整的外膜蛋白PagL的核磁共振研究。我们比较了均匀的13C，15N标记的WC的13C和15NCP-MAS谱与从PagL高产的大肠杆菌细胞中分离的CE，在存在和不存在微波辐射的情况下。在较高的温度(271K)下，E.coliCE的单核磁共振谱先前已经揭示了PagL和内源膜相关大分子的原子细节，包括主要的脂蛋白lpp和非蛋白质成分，如脂多糖、肽多聚(PG)和磷脂。[3]在低温(LT)dnp条件下，我们在蛋白质信号的特征光谱区域(脂肪族13C共振：δ=50-55ppm，酰胺15N主链和侧链共振在约120和80-30ppm)中观察到两种制剂的显著DNP增强因子。
Solid-state NMR (ssNMR) spectroscopy offers increasing possibilities to study complex biomolecules at the atomic level.[1] An important target area concerns membrane-associated proteins, which can be investigated by ssNMR methods after reconstitution in synthetic bilayers. While such preparations allow examination of functional aspects of the protein of interest, the influence of the native cellular environment on protein structure and function cannot be monitored. Very recently, we introduced a general approach aimed at determining complex molecular structures, including integral membrane proteins, in their native cellular environment by ssNMR under magic-angle-spinning (MAS) conditions.[2, 3] Using dedicated sample-preparation routes, we demonstrated that high-resolution ssNMR spectra can be obtained on uniformly 13C, 15N-labeled preparations of Escherichia coli whole cells (WC) and cell envelopes (CE). Both CE and WC morphology are preserved under standard ssNMR experimental conditions and the corresponding 13C and 15N crosspolarization (CP-MAS) spectra are invariant over time. However, with increasing levels of molecular complexity, especially in the case of WC preparations, spectroscopic sensitivity becomes a critical factor. In recent years, dynamic nuclear polarization (DNP) has developed into a routine tool to increase the sensitivity of multidimensional ssNMR.[4] DNP enhancements of up to 148-fold have been obtained on micro/nanocrystalline biomolecular samples, including an amyloidogenic peptide and a deuterated protein,[5, 6] while enhancements between 18-and 46-fold have been reported for membrane-embedded polypeptides, purple membrane preparations, and bacteriophages.[7, 8] Here, we investigated the use of DNP to conduct ssNMR studies on 13C, 15N-labeled preparations of E. coli WC overproducing the integral outer membrane protein PagL.[9] InFigure1, we compared 13C and 15NCP-MAS spectra of uniformly 13C, 15N-labeled WC with the CE isolated from PagL-overproducing E. coli cells, recorded in the presence and absence of microwave irradiation. At higher temperatures (271K), ssNMR spectra of the E. coli CE had previously revealed atomic details of PagL as well as endogenous membrane-associated macromolecules, including the major lipoprotein Lpp and non-proteinaceous components such as lipopolysaccharides (LPS), peptidoglycans (PG), and phospholipids.[3] Under low-temperature (LT) DNP conditions, we observed significant DNP enhancement factors for both preparations in spectral regions characteristic for protein signals (aliphatic 13C resonances: δ= 50–55 ppm, amide 15N backbone and side-chain resonances at about 120 and 80–30ppm) as well as for 13C signals of endogenous