Development of Solid-State NMR Method for Studying Higher-Order Structure of Proteins

研究蛋白质高阶结构的固态核磁共振方法的发展

• 批准号: 09558091
• 负责人: FUJIWARA Toshimichi
• 金额: $ 8.51万
• 依托单位: Yokohama National University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09558091/
• 关键词: Solid-State NMR; Labeling with Stable Isotope; Analysis of Protein Higher-Order Structure; Structural Biology; 二面角; 生体分子構造; タンパク質; 結合長; 結合角; 多次元NMR

Multi-dimensional correlation NMR has been developed especially for ィイD113ィエD1C, ィイD115ィエD1N labeled biomolecules in solid states. Multi-dimensional NMR that has two or three isotropic shift evolution periods under magic-angle spinning conditions resolve ィイD113ィエD1C and ィイD115ィエD1N signals of uniformly isotope-labeled biomolecules. Dipolar couplings for ィイD113ィエD1C and ィイD115ィエD1 spins in labeled molecules provide spin connectivity for signal assignment and internuclear distances information for the structure determination. Analysis of the mutual orientation of anisotropic interactions for ィイD113ィエD1C and ィイD115ィエD1N spins such as chemical shift anisotropies and dipolar interactions permit the determination of dihedral angles.Dihedral angles can be determined from the correlations between anisotropic interactions of different spins, because such correlation spectra depend on mutual orientations of anisotropic interactions which specify dihedral angles. These correlation spectra were ob … More tained with experiments in which evolutions of anisotropic interactions are connected by a dipolar mixing period. This experiment is combined with the isotropic-shift correlation to enhance the spectral resolution. Example are shown for the correlation of the ィイD113ィエD1CィイD11ィエD1H and ィイD113ィエD1CィイD113ィエD1C dipolar couplings in an ィイD11ィエD1HィイD1α13ィエD1CィイD1αィエD1-ィイD113ィエD1CィイD1β1ィエD1HィイD1βィエD1 moiety and for the correlation of the ィイD113ィエD1CO chemical shift anisotropy and CH dipolar interactions. The experimental pulse sequence includes ィイD113ィエD1CィイD11ィエD1H dipolar evolution periods for ィイD113ィエD1CィイD1αィエD1 and ィイD113ィエD1CィイD1βィエD1H, which are correlated with a coherent ィイD113ィエD1CィイD1α13ィエD1CィイD1βィエD1 dipolar mixing period. This method enables the experimental determination of all the structural parameters for the four-spin system.A pulse sequence applied synchronously with sample-spinning is designed for enhancing magnetization transfer between ィイD113ィエD1C spins. This method improve the transfer efficiency by reducing γ-dependence of the zero-quantum dipolar mixing operator. This efficiency was verified for fully ィイD113ィエD1C labeled alanine.Sample preparation for solid state NMR has also been improved. Rapid sample cooling and cooling with protectant such as trehalose reduce the signal broadening due to distribution of molecular conformations. This method was applied to ATP complexed with FィイD21ィエD2-ATP synthase β subunit. The conformation was monitored with ィイD131ィエD1P-NMR. Less

多维相关核磁共振技术是近年来发展起来的一种用于固体状态下生物分子标记的核磁共振技术。在魔角旋转条件下具有两个或三个各向同性位移演化周期的多维NMR解析均匀同位素标记的生物分子的D113-D1C和D115-D1 N信号。偶极耦合标记的分子中的D113的D1C和D115的D1自旋提供自旋连接的信号分配和核间距离的信息，用于结构确定。通过分析自旋为D113的各向异性D1C和D115的各向异性D1 N自旋的相互作用的取向，如化学位移各向异性和偶极相互作用，可以确定二面角。二面角可以从不同自旋的各向异性相互作用之间的相关性来确定，因为这种相关谱依赖于指定二面角的各向异性相互作用的相互取向。这些相关谱是由 ...更多信息 得到的实验中，各向异性相互作用的演变连接的偶极混合期。该实验结合各向同性位移相关来提高光谱分辨率。示例显示了在一个环D11环D1 H环D1 H环D1α13环D1C环D1α环D1-环D113环D1C环D1β1环D1 H环D1 H和环D113环D1C环D1C环D113环D1C偶极耦合的相关性，以及环D113环D1CO化学位移各向异性和CH偶极相互作用的相关性。实验脉冲序列包括D113 D1C D11 D1 H偶极演化周期，D113 D1C D1α D1 H和D113 D1C D1β D1 H偶极演化周期与相干D113 D1C D1α13 D1C D1β D1偶极混合周期相关。设计了一个与样品自旋同步的脉冲序列，用于增强双自旋D113双自旋D1C自旋之间的磁化转移。该方法通过降低零量子偶极混合算符的γ依赖性来提高传输效率。该方法在全反式D113双反式D1C标记的丙氨酸中得到了验证，并改进了固体核磁共振的样品制备。快速样品冷却和用保护剂如海藻糖冷却可减少由于分子构象分布引起的信号展宽。该方法适用于ATP与F_（12）D_2-ATP合酶β亚基复合物的分析。构象用1H NMR D131核磁共振D1 P-NMR监测。少

项目成果

Toshimichi Fujiwara: "Multidimensional Solid-State Nuclear Magnetic Resonance for Determining the Dihedral Angle from the Correlation of ^<13>C-^1H and ^<13>C-^<13>C Dipolar Interactions under Magic-Angle Spinning Conditions" Journal of Chemical Physics.

Toshimichi Fujiwara：“多维固态核磁共振根据魔角旋转条件下 ^<13>C-^1H 和 ^<13>C-^<13>C 偶极相互作用的相关性确定二面角”期刊

Toshimichi Fujiwara: "Multidimensional Solid-State Nuclear Magnetic Resonance for Correlating Anisotropic Interactionss under Magic-Angle Spinning Conditions"J. Magn. Reson.. 124. 147-153 (1997)

Toshimichi Fujiwara：“用于关联魔角旋转条件下各向异性相互作用的多维固态核磁共振”J。

Sumie Yoshioka: "Molecular Mobility of Protein in Lyophilized Formulations Linked to the Molecular Mobility of Polymer Excipients, as Determined by High Resolution ^<13>C Solid-State NMR"Pharmaceutical Research. 16. 1621-1625 (1999)

Sumie Yoshioka：“冻干制剂中蛋白质的分子迁移率与聚合物赋形剂的分子迁移率相关，通过高分辨率13 C固态NMR测定”药物研究。

Toshimichi Fujiwara: "Multidimensional Solid-State Nuclear Magnetic Resonance for Correlating Anisotropic Interactions under Magic-Angle Spinning Conditions" Journal of Magnetic Resonance. 124. 147-153 (1997)

Toshimichi Fujiwara：“用于关联魔角旋转条件下各向异性相互作用的多维固态核磁共振”磁共振杂志。

Suhkman Kim: "The Interactions of Ferric and Ferrous Cytochrome c with Cardiolipin in Phospholipid Membranes Studied by Solid-State ィイD12ィエD1H and ィイD131ィエD1P NMR"J. Mol. Struct.. 441. 147-153 (1998)

Suhkman Kim：“通过固态 D12D1H 和 D131D1P NMR 研究三价铁和亚铁细胞色素 c 与磷脂膜中的心磷脂的相互作用”J. 441. 147-153 (1998)