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

这项研究试图开发一种方法来精确和准确地确定生物分子特定局部区域中的碳-碳和碳-氮距离以及二面角phi、psi。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抑制素(SSI)和枯草杆菌菌素的复合体。获得了对理解SSI抑制机制至关重要的结构数据。1.我们提出的方法，MLEV-4，4‘，MLEV8/8^R，RACO和R2TR，被进一步发展为实用工具。2.发展了一个通过调制应用于自旋的射频场的幅度、相位和频率来操纵内部哈密顿量的理论。3.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。4.制备了链霉菌枯草菌素抑制剂(SSI)与枯草杆菌菌素的络合物，其中剪切键上的碳和氮选择性地被它们的同位素(^&lt；13&gt；C和^&lt；15&gt；N)所取代精确测定了标记的SSI-枯草杆菌素络合物样品的15&gt；N距离，并推导出该络合物标记区的加速结构。6.将测定的结构与X射线衍射法得到的结构进行比较，探讨其缓蚀机理。

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.