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Development of solid NMR methods for structural determination of a peptide

开发用于肽结构测定的固体核磁共振方法

基本信息

批准号：
13558081
负责人：
TERAO Takehiko
金额：
$ 5.76万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13558081/
关键词：
solid NMR structural determination peptide dihedral angle nuclear distance measurements

项目摘要

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

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整的3D结构的13 <13>C-标记的生物分子。在第一年，我们开发了一种新的方法，并修改了我们的NMR机器如下：（1）^1H-^<13>C偶极辅助旋转共振（DARR）的开发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射，因此距离测量的混合时间受到通常较长的自旋-晶格弛豫时间的限制。这使我们能够测量更长的距离。此外，我们表明，DARR有许多特点，适用于应用到一个多同位素标记的样品，如，观察是unedr高分辨率条件下进行。(2)低温观测装置的安装为了准确测定分子结构，通过低温抑制局部分子运动 ...更多信息设定样品温度是先决条件。我们购买了一个新开发的低温核磁共振观测系统和一个液氮再冷凝系统。这些系统与我们的NMR系统相结合，实现了安全稳定的长时间低温实验。第二年，我们应用DARR方法确定了神经递质-用<13><15>固相合成法合成了一个全标记的肽样品，它由6个残基组成（K-F-I-G-L-M），是速激肽家族的共同C-末端序列。用DARR进行2D ^<13>C-^<13>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，并且基于81个距离约束成功地获得了3D结构。此外，确定肽主链的四个φ二面角，并将其作为角度约束输入X-Plor程序。结果表明，通过增加这些二面角约束，结构的精度得到了提高。总之，在本项目中，我们可以建立固体NMR方法，使我们能够通过使用一个多重标记的肽样品的一些2D固体NMR实验，以合理的准确度确定肽分子的3D结构。少