Development of solid NMR methods for structural determination of a peptide

本研究的目的是建立一种固体核磁共振方法，使我们能够确定一个完全^<13> 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以下改进：(1)开发了^1H-^<13>C偶极辅助旋转共振（DARR）我们开发了一种方法，使我们能够通过一个二维实验确定大量的距离。这种方法一直是人们所期望的，但尚未实现。由于DARR没有对观察到的自旋施加射频辐照，因此用于距离测量的混合时间通常受到长自旋晶格弛豫时间的限制。这使我们能够测量更远的距离。此外，我们发现DARR具有许多适合应用于多重同位素标记样品的特征，例如，在高分辨率条件下进行观察。(2)安装低温观测装置对于分子结构的累积测定，通过降低温度来抑制局部分子运动是先决条件。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。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时间低温实验。在第二年，我们应用了DARR方法来确定eledoisin相关肽的结构，这是一种神经递质。采用固体合成法合成了一个由速激肽家族常见的C端6个残基（K-F-I-G-L-M）组成的完全^<13>C和^<15>N标记的肽样品。结构测定方法如下：用DARR进行2D ^<13>C-^<13>C距离相关实验，得到81个相关C-C距离。这些距离被输入到耶鲁大学开发的X-Plor程序中，并成功地获得了基于81个距离约束的3D结构。此外，确定了肽主链的4个φ二面角，并将其作为角度约束输入X-Plor程序。结果表明，加入这些二面角约束后，结构精度得到了提高。综上所述，在本项目中，我们可以建立固体核磁共振方法，使我们能够通过少量的二维固体核磁共振实验，使用一个多重标记的肽样品，以合理的精度确定肽分子的三维结构。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