Sensitivity-enhanced NMR of biological solids: Dynamic nuclear polarization of Y21M fd bacteriophage and purple membrane
Sensitivity-enhanced NMR of biological solids: Dynamic nuclear polarization of Y21M fd bacteriophage and purple membrane
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DOI：
10.1021/ja005659j
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发表时间：
2001-02-07
影响因子：
15
通讯作者：
Griffin, RG
中科院分区：
文献类型：
--
作者：
Rosay, M;Zeri, AC;Griffin, RG
固体核磁共振（SSNMR）已成为从生物和化学体系中提取结构参数的有力工具。然而，核磁共振固有的低灵敏度往往限制了结构研究的范围。动态核极化（DNP）可以解决这个问题，因为信号强度可以提高2到3个数量级。这种灵敏度的增益是通过在EPR拉莫尔频率或附近的微波辐射将外源顺磁分子中的电子的极化转移到核自旋来实现的。DNP信号增强的大小取决于许多因素，包括核自旋晶格弛豫时间T1，该时间在低温下进行实验时达到最大。具有丰富甲基的系统，如病毒颗粒和膜蛋白，即使在低温下也通常具有较短的T1，并且可能是DNP实验的特别具有挑战性的候选者。然而，这些也是SSNMR结构研究中最有趣的一些案例，因为它们通常不能用溶液核磁共振或x射线衍射来检查。在这篇文章中，我们证明了有效极化上述每个系统的原型例子是可能的，即病毒颗粒fd和含有细菌视紫红质（bR）及其伴随的脂质的紫色膜。此外，通过比较fd噬菌体的15N和31P光谱中的DNP信号增强，我们发现1H自旋扩散将增强的极化均匀分布在整个大分子组装体中。这些结果表明DNP可能是一种普遍适用于SSNMR实验灵敏度增强的方法。我们一直在开发用于高磁场研究的DNP技术[最初在5 T，最近在9 T（140和250 GHz EPR以及211和380 MHz 1H NMR频率）]。到目前为止，我们最成功的极化转移实验是基于热混合5,6与氮氧自由基4-氨基- tempo掺杂到水/甘油。这个实验很简单
Solid-state NMR (SSNMR) is established as a powerful tool for extracting structural parameters from biological and chemical systems. 1-3 However, the inherent low sensitivity of NMR often limits the scope of structural studies. With dynamic nuclear polarization (DNP) it is possible to address this issue since signal intensities can be enhanced by 2 to 3 orders of magnitude. This gain in sensitivity is accomplished by transferring polarization from electrons in exogenous paramagnetic molecules to nuclear spins via microwave irradiation at or near the EPR Larmor frequency. 4 The size of the DNP signal enhancement is dependent on a number of factors including the nuclear spin lattice relaxation time, T1, which is maximized by performing experiments at low temperatures. Systems with an abundance of methyl groups, such as virus particles and membrane proteins, often have short T1’s even at low temperatures and could be particularly challenging candidates for DNP experiments. However, these are also some of the most interesting cases for SSNMR structural studies since they often cannot be examined with either solution NMR or X-ray diffraction. In this communication we demonstrate that it is possible to efficiently polarize archetypal examples of each of the systems mentioned above-namely the viral particle fd and the purple membrane containing bacteriorhodopsin (bR) and its accompanying lipids. Furthermore, by comparing the DNP signal enhancements in the 15N and 31P spectra of fd bacteriophage, we show that 1H spin diffusion evenly distributes the enhanced polarization throughout a large macromolecular assembly. These results suggest that DNP may be a generally applicable approach for sensitivity enhancement in SSNMR experiments.We have been developing DNP techniques for studies at high magnetic fields [initially at 5 T and more recently at 9 T (140 and 250 GHz EPR and 211 and 380 MHz 1H NMR frequencies)]. To date, our most successful polarization transfer experiments are based on thermal mixing5, 6 with the nitroxide radical 4-amino-TEMPO doped into water/glycerol. The experiment is imple-