Rosay Melanie, Lansing Jonathan C, Haddad Kristin C, Bachovchin William W, Herzfeld Judith, Temkin Richard J, Griffin Robert G
Francis Bitter Magnet Laboratory and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Am Chem Soc. 2003 Nov 12;125(45):13626-7. doi: 10.1021/ja036898k.
One of the principal promises of solid-state NMR (SSNMR) magic angle spinning (MAS) experiments has been the possibility of determining the structures of molecules in states that are not accessible via X-ray or solution NMR experiments-e.g., membrane or amyloid proteins. However, the low sensitivity of SSNMR often restricts structural studies to small-model compounds and precludes many higher-dimensional solid-state MAS experiments on such systems. To address the sensitivity problem, we have developed experiments that utilize dynamic nuclear polarization (DNP) to enhance sensitivity. In this communication, we report the successful application of MAS DNP to samples of cryoprotected soluble and membrane proteins. In particular, we have observed DNP signal enhancements of up to 50 in 15N MAS spectra of bacteriorhodopsin (bR) and alpha-lytic protease (alpha-LP). The spectra were recorded at approximately 90 K where MAS is experimentally straightforward, and the results suggest that the described protocol will be widely applicable.
固态核磁共振（SSNMR）魔角旋转（MAS）实验的一个主要前景是，有可能确定那些无法通过X射线或溶液核磁共振实验得到的分子结构，例如膜蛋白或淀粉样蛋白。然而，SSNMR的低灵敏度常常将结构研究限制在小分子模型化合物上，并且排除了在此类体系上进行许多更高维度的固态MAS实验。为了解决灵敏度问题，我们开发了利用动态核极化（DNP）来提高灵敏度的实验。在本通讯中，我们报告了MAS DNP在冷冻保护的可溶性蛋白和膜蛋白样品上的成功应用。特别是，我们在细菌视紫红质（bR）和α-溶菌蛋白酶（α-LP）的15N MAS谱中观察到了高达50倍的DNP信号增强。这些谱图是在约90 K下记录的，此时MAS在实验上很简便，结果表明所描述的方案将得到广泛应用。
