Development of solid NMR methods for structural determination of a peptide

这项研究的目的是建立一种固体核磁共振方法，使我们能够确定完全^&lt；13&gt；C标记的生物分子的三维结构。在第一年，我们开发了一种新的方法，并对我们的核磁共振机进行了如下改进：(1)研制了偶极辅助旋转共振(DAR)。我们开发了一种方法，使我们能够通过一个2D实验来确定大量的距离。这种方法长期以来一直是人们所希望的，但尚未实现。由于Darr deos不对观测到的自旋进行射频辐照，用于距离测量的混合时间通常受到较长的自旋-晶格弛豫时间的限制。这使我们能够测量更长的距离。此外，我们还证明了DAR具有许多适合于多同位素标记样品的特点，例如，在高分辨率条件下进行观察。(2)安装低温观测装置为了准确地确定分子结构，通过较低的…抑制局部分子运动更高的样品温度是先决条件。我们购买了最近开发的低温核磁共振观测系统和液氮再冷凝系统。将这些系统与我们的核磁共振系统相结合，实现了安全稳定的长期低温实验。第二年，我们应用DAR方法确定了作为神经递质的肾上腺素相关肽的结构。用固相合成法合成了速激肽家族常见的C末端序列K-F-I-G-L-M组成的完整的^&lt；13&gt；C和^&lt；15&gt；N标记肽样品。结构测定如下：通过与DarR的2D^&lt；13&gt；C-^&lt；13&gt；C距离相关实验，得到81个相关的C-C距离。这些距离被输入到耶鲁大学开发的X-PLOR程序中，并基于81个距离约束成功地获得了3D结构。进一步，确定了多肽主链的四个φ二面角，并作为角度约束输入到X-PLOR程序中。结果表明，加入这些二面角约束后，结构的精度得到了提高。综上所述，在本项目中，我们可以建立固体核磁共振方法，使我们能够通过使用一个多标记多肽样品进行少量的2D固体核磁共振实验，以合理的精度确定多肽分子的三维结构。较少

The aim of this research is to establish a solid-NMR method, which enables us to determine a 3D structure of a fully ^<13>C-labeled biomolecule. In the first year, we developed a new method and modified our NMR machine as follows:(1) Development of ^1H-^<13>C dipolar assisted rotational resonance (DARR)We developed a method enabling us to determine a large number of distances by one 2D experiment. Such method has long been desired but not yet been realized. Since DARR deos not apply rf irradiation to the observed spins, the mixing time for distance measurement is limited by generally long spin-lattice relaxation times. This enables us to measure longer distances. Further, we showed that DARR has many features suitable for application to a multiply isotope-labeled sample, such as, observation is done unedr high-resolition conditions.(2) Installation of low-temperature observation apparatusFor acculate determination of a molecular structure, suppression of local molecular motion by lower … More ing the sample temperature is prerequisite. We bought a recently developed low temperature NMR observation system and also a liquid-nitrogen recondensation system. These systems were combined with our NMR system to realize safe and stable long low-temperature experiments.In the second year, we applied the DARR method to determine the structure of the eledoisin-related peptide which is a neurotransmitter. A fully ^<13>C and ^<15>N labeled peptide sample conposed of six residues (K-F-I-G-L-M), which is the common C-terminal sequence of the tachykinin family, was synthesized by using a solid synthesis procedure. The structural determination was done as follows, 81 relevant C-C distances were obtaned from the 2D ^<13>C-^<13>C distance-correlation experiment with DARR. These distances were fed into the X-Plor program developed at Yale University and a 3D structure was successfully obtaned based on the 81 distance constraines. Further, the four φ dihedral angles of the peptide main chain were determined and fed also into the X-Plor program as angle constrains. It was shown that the precision of the structure becomes better by adding these dihedral angle constraints. To summarize, in this project, we can establish the solid-NMR method enabling us to determine a 3D structure of a peptide molecule with reasonable acculacy by a few numbers of 2D solid-NMR experiments using one multiply labeled peptide sample. Less