Lopez Jakob J, Kaiser Christoph, Shastri Sarika, Glaubitz Clemens
Institute for Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J. W. Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany.
J Biomol NMR. 2008 Jun;41(2):97-104. doi: 10.1007/s10858-008-9245-3. Epub 2008 May 28.
13C homonuclear correlation spectra based on proton driven spin diffusion (PDSD) are becoming increasingly important for obtaining distance constraints from multiply labeled biomolecules by MAS NMR. One particular challenging situation arises when such constraints are to be obtained from spectra with a large natural abundance signal background which causes detrimental diagonal peak intensities. They obscure cross peaks, and furthermore impede the calculation of a buildup rates matrix which may be used to derive distance constraints, as carried out in "NMR crystallography". Here, we combine double quantum (DQ) filtering with 13C-13C dipolar assisted rotational resonance (DARR) experiments to yield correlation spectra free of natural abundance contributions. Two experimental schemes, using DQ filtering prior to evolution (DOPE), and after mixing (DOAM), have been evaluated. Diagonal peak intensities along the spectrum diagonal are removed completely, and crosspeaks close to the diagonal are easily identifiable. For DOAM spectra with negligible mixing times, it is possible to carry out 'assignment walks' which simplify peak identification substantially. The method is demonstrated on 13C-cys labeled proteorhodopsin, a 27 kDa membrane protein. The magnetization transfer characteristics were studied using buildup curves obtained on uniformly 13C labelled crystalline tripeptide MLF. Our data show that DQ filtered DARR experiments pave the way for obtaining through space constraints for structural studies on ligands, bound to membrane receptors, or on small fragments within large proteins.
基于质子驱动自旋扩散（PDSD）的13C同核相关谱对于通过MAS NMR从多重标记的生物分子中获取距离约束变得越来越重要。当要从具有大量天然丰度信号背景的谱中获得此类约束时，会出现一种特别具有挑战性的情况，这种背景会导致有害的对角峰强度。它们会掩盖交叉峰，并且还会妨碍构建速率矩阵的计算，而该矩阵可用于推导距离约束，如在“NMR晶体学”中所进行的那样。在这里，我们将双量子（DQ）滤波与13C-13C偶极辅助旋转共振（DARR）实验相结合，以产生不含天然丰度贡献的相关谱。已经评估了两种实验方案，即在演化之前使用DQ滤波（DOPE）和在混合之后使用DQ滤波（DOAM）。沿着谱对角线的对角峰强度被完全去除，并且靠近对角线的交叉峰很容易识别。对于混合时间可忽略不计的DOAM谱，可以进行“归属游走”，这大大简化了峰的识别。该方法在13C-半胱氨酸标记的视紫质（一种27 kDa的膜蛋白）上得到了验证。使用在均匀13C标记的结晶三肽MLF上获得的积累曲线研究了磁化转移特性。我们的数据表明，DQ滤波的DARR实验为获得空间约束铺平了道路，可用于对与膜受体结合的配体或大蛋白质内的小片段进行结构研究。
