Precise determination of local structures of biomolecules by solid-state NMR

本研究试图开发精确和准确测定碳-碳和碳-氮距离以及二面角phi， psi在生物分子的特定局部区域的方法。它的目的是应用于生物学中尚未被x射线晶体学或溶液态核磁共振解决的结构问题。在这一年里，我们详细阐述了我们开发的几种方法，并将它们应用于枯草杆菌链霉菌抑制剂（SSI）和枯草杆菌素的复合物。获得了对了解SSI抑制剂机制至关重要的结构数据1。我们提出的mlev -4,4', MLEV8/8^R， RACO和R2TR方法得到了进一步的发展，作为实用的工具。一个理论，这是至关重要的操纵内部哈密顿量通过调制振幅，相位和频率的射频场应用于自旋，发展。该方法成功地恢复了快速魔角旋转下^<13>C-^<15>N偶极体粉末的各向异性化学位移。从理论上考察了晶格振动对x射线衍射和固体核磁共振测量距离的影响。制备了枯草链霉菌抑制剂（SSI）与枯草杆菌素的配合物，其中碳和氮在可剪键上被选择性地替换为它们的同位素（^<13>C和^<15>N） 5。精确测定了标记的ssi -枯草菌素配合物样品的^<13>C-^<15>N距离，并推导出配合物标记区域的加速结构。将上述所得结构与x射线衍射所得结构进行比较，探讨缓蚀剂的机理。

This study attempted to develop methods for precise and accurate determination of carbon-carbon and carbon-nitrogen distances as well as dihedral angles phi, psi in a particular local region of a biomolecule. It was aimed at applying to structural problems in a biology which have yet not been solved by either X-ray crystallography or solution-state NMR.In this year, we elaborated several methods which we had developed, and applied them to the complex of streptomyces subtilisin inhibitor (SSI) and subtilisin. Structural dada, which is essential for understanding the inhibitor mechanism of SSI,was obtained1.The methods which we had proposed, MLEV-4,4', MLEV8/8^R, RACO,and R2TR,were further developed to be used as practical tools.2.A theory, which is vital to manipulate internal Hamiltonians by modulating amplitudes, phases, and frequencies of a radio-frequency field applied to spins, was developed. This method was applied successfully to recover anisotropic chemical shift or ^<13>C-^<15>N dipolar powder patterns under fast magic angle spinning.3.Lattice vibration effects on distances measured by X-ray diffraction and solid-state NMR were theoretically examined.4.The complex of streptomyces subtilisin inhibitor (SSI) and subtilisin was prepared, where the carbon and the nitrogen at the scissile bond were selectively replaced by their isotopes (^<13>C and ^<15>N).5.The ^<13>C-^<15>N distance of the labeled SSI-subtilisin complex sample was precisely determinded, and an accelerate structure of the labeled region of the complex was deduced.6.The structure determined as above and that obtained by X-ray diffraction were compared to discuss the inhibitor mechanism.