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

Multi-dimensional correlation NMR has been developed especially for I D 113个D 1C，I D 115个D 1 N labeled biomolecules in solid states.Multi-dimensional NMR that has two or three isotropic shift evolution periods under magic-angle spinning conditions resolve Ney D113个D1C and I D115个D1N signals of uniformly isotope-labeled biomolecules。Dipolar couplings for I D 113个D 1C and I D 115个D 1 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 I D 113个文件D1C and I D 115个文件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 which specify hedr.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 I D113文件D1C I D 11文件D1H and I D 113文件D 1C I D 113文件D 1C dipolar couplings in an I D 11文件D 1 H文件D1α13文件D 1 C文件D 1α文件D 1-I D 113文件D 1 C文件D 1β1文件D 1 H文件D 1βD 1 moiety and for the correlation of the I D 113文件D 1 CO chemical shift anisotropy and CH dipolar interactions.The experimental pulse sequence includes I D113文件D1C文件D11文件D1H dipolar evolution periods for I D113文件D1C文件D1α文件D1 and文件D113文件D1C文件D1C文件D1β文件D1H，which are correlated with a coherent文件D113文件D1C文件D1α13文件D1C文件D1β文件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 y D113文件D1C spins.This method improve the transfer efficiency by reducingγ-dependence of the zero-quantum dipolar mixing operator.This efficiency was verified for fully y D113文件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 I D21 ii D2-ATP synthaseβsubunit.The conformation was monitored with I D131 I D 1 P-NMR。Less：Less

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