Relaxation-compensated difference spin diffusion NMR for detecting 13C-13C long-range correlations in proteins and polysaccharides.
Relaxation-compensated difference spin diffusion NMR for detecting 13C-13C long-range correlations in proteins and polysaccharides.
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DOI：
10.1007/s10858-014-9889-0
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发表时间：
2015-02
影响因子：
2.7
通讯作者：
Hong M
中科院分区：
文献类型：
--
作者：
Wang T;Williams JK;Schmidt-Rohr K;Hong M
在生物大分子的固态核磁共振结构测定中，长程距离的测量仍然是一个挑战。在均匀¹³C标记的生物分子的二维和三维相关谱中，为三维结构提供重要长程距离约束的残基间、片段间和分子间的¹³C - ¹³C交叉峰常常与仅反映分子共价结构的短程交叉峰重叠。因此，开发仅获得长程交叉峰的新方法是可取的。在此我们表明，对常用的二维¹H驱动自旋扩散（PDSD）实验进行弛豫补偿改进，可以清晰地检测到此类长程交叉峰。通过添加一个z - 滤波器以保持实验的总z周期恒定，我们补偿了¹³C T₁弛豫。结果，长混合时间谱和按比例缩放的短混合时间谱之间的差谱仅显示长程相关信号。我们表明，一键和二键交叉峰在几十毫秒内达到平衡。在约200毫秒内，氨基酸残基和单糖内的强度平衡到一个反映局部网络中自旋数量的值。通过T₁弛豫补偿，在更长的混合时间下，残基间和片段间交叉峰强度增加，而片段内交叉峰强度相对于彼此保持不变，并且都可以被扣除。没有弛豫补偿时，由于大多数生物分子中T₁弛豫的不均匀性，二维差谱会同时出现正负强度，这可能导致峰的抵消。我们在氨基酸、单糖、一种晶体模型肽、一种膜结合肽和一种植物细胞壁样品上展示了这种弛豫补偿差PDSD方法。所得的差谱产生清晰的多键、残基间和分子间相关峰，这些峰在原始二维谱中通常难以分辨。
The measurement of long-range distances remains a challenge in solid-state NMR structure determination of biological macromolecules. In 2D and 3D correlation spectra of uniformly 13C-labeled biomolecules, inter-residue, inter-segmental, and intermolecular 13C-13C cross peaks that provide important long-range distance constraints for three-dimensional structures often overlap with short-range cross peaks that only reflect the covalent structure of the molecule. It is therefore desirable to develop new approaches to obtain spectra containing only long-range cross peaks. Here we show that a relaxation-compensated modification of the commonly used 2D 1H-driven spin diffusion (PDSD) experiment allows the clean detection of such long-range cross peaks. By adding a z-filter to keep the total z-period of the experiment constant, we compensate for 13C T1 relaxation. As a result, the difference spectrum between a long- and a scaled short-mixing time spectrum show only long-range correlation signals. We show that one- and two-bond cross peaks equalize within a few tens of milliseconds. Within ~200 ms, the intensity equilibrates within an amino acid residue and a monosaccharide to a value that reflects the number of spins in the local network. With T1 relaxation compensation, at longer mixing times, inter-residue and inter-segmental cross peaks increase in intensity whereas intra-segmental cross-peak intensities remain unchanged relative to each other and can all be subtracted out. Without relaxation compensation, the difference 2D spectra exhibit both negative and positive intensities due to heterogeneous T1 relaxation in most biomolecules, which can cause peak cancellation. We demonstrate this relaxation-compensated difference PDSD approach on amino acids, monosaccharides, a crystalline model peptide, a membrane-bound peptide and a plant cell wall sample. The resulting difference spectra yield clean multi-bond, inter-residue and intermolecular correlation peaks, which are often difficult to resolve in the parent 2D spectra.