SOLID STATE NMR MEASURE OF PINPEPTIDES AN NCCN2Q HETERONUCLEAR LOCAL FIELD

本文介绍了一种固体核磁共振实验， （15 N-13 C ~-13 C 0- 15 N）标记肽的扭转角。 创建 13 Cu-13 C o双量子相干（DQC）之后是选择性的 在15 N- 3C偶极相互作用下的演化（Costa，1997; Feng， 1997年）。 ~ 3Cc~- 3C o DQC失相的时间过程对 P扭转角，特别是与B片相关的值 二级结构（l20 o ~ l 80 o）。 我们将这项技术应用于 甘氨酰甘氨酸盐酸盐（GG），并提取与 衍射测定值（P= 162.10），精度为q5 g。 Feng，et.例如，最近提出了一种新颖的， 基于MAS的13 CH 1 3CH键扭转角测量方法 （表示为2 Q-异核局域场（HLF））通过检查相对 两个直接键合的13 C-1H偶极相互作用的取向 (Feng，1996）。 对15 NH-13 CH扭转也提出了类似的建议 angles（Hong，1997）. 这些实验是一种通用的 空间各向异性自旋相关的一类测量 偶极耦合和化学位移等相互作用 各向异性（CSA）来提取结构信息。 因为这些 技术关联大自旋相互作用，它们更容易 扩展到多重标记的样本，而弱核间 偶极相互作用，其大小提供有用的距离 信息可能会被强耦合所掩盖， 直接键合的原子核。 在这里，我们描述了2 Q-HLF的修改 15 N-1 3C-1 3C-1 5 N（NCCN）自旋四重态。 创作 13 C2 DQC的影响下，随后仅在13 C2 DQC的影响下失相。 15 N-13 C异源耦合，将产生DQC退相曲线 其形状提供了关于 15 N-13 C联轴器，因此是“3 C-13 C”扭转角。 这 对应于当13 C与13 C结合时沿着肽主链的“-P角”。 核是直接键合的Cx-和羰基碳。 通过重新耦合 在退相期间的'5 N-13 C相互作用，而不是 在（非常）慢的单个转子周期期间观察它们的影响 旋转速度，我们能够有效地进行实验， 更高的纺丝速度和更精细的时间分辨率。 的 实验结果表明，至少在一定的扭转角下， 制度，是非常敏感的构象。 因为13 C =O 缺乏结合的1H，这代表了一种独特的方法（缺少17 O NMR） 应用2 Q-HLF概念测量肽骨架~P 的角度 这提供了一个测量肽的一般框架 涉及13 C-15 N偶极的固体中的主链扭转角tP 13 Ccx-13 C o DQ相干性的退相。 具体实施 这里所描述的是设计成在相对高的温度下有效地工作。 旋转速度 对构象高度敏感的区域（~P= 12 O- 1800）大致对应于B片结构区。 这种二级结构的延伸性质使其 很难准确定义肽的结构细节， 这是使用距离测量技术进行的， 5-6 A。NCCN 2 Q-HLF技术在这些系统中的应用 应该是特别有用的，在划定的确切性质， 它们的B折叠构象。

A solid-state NMR experiment has been described for measuring P torsion angles in (15N-13C~-13C 0-15N)-labeled peptides. Creation of 13Cu-13C o double-quantum coherence (DQC) is followed by selective evolution under '5N- 3C dipolar interactions (Costa, 1997; Feng, 1997). The time-course of '3Cc~- 3C o DQC dephasing is sensitive to the P torsion angle, particularly for values associated with B-sheet secondary structure (l20o~l80o). We apply the technique to glycylglycineHCl (GG) and extract a value for P that matches the diffraction-determined value (P= 162.10) with a precision of q5g. Feng, et. al., have recently proposed a novel, magic angle spinning (MAS)-based method for measuring torsion angles about 13CH1 3CH bonds (denoted 2Q-Heteronuclear Local Field (HLF)) by examining the relative orientation of the two directly-bonded 13C-1H dipolar interactions (Feng, 1996). A similar proposal has been made for 15NH-13CH torsion angles (Hong, 1997). These experiments are examples of a general class of measurements which correlate spatially anisotropic spin interactions such as the dipolar coupling and the chemical shift anisotropy (CSA) to extract structural information. Because these techniques correlate large spin interactions, they are more easily extended to multiply-labeled samples, whereas the weak internuclear dipolar interactions whose magnitudes provide useful distance information may be obscured by strong couplings between directly-bonded nuclei. Here we describe a modification of the 2Q-HLF concept applied to a 15N-1 3C-1 3C-1 5N (NCCN) spin quartet. Creation of 13C2 DQC, followed by dephasing solely under the influence of the 15N-13C heteronuclear couplings, will yield a DQC dephasing curve whose shape provides information about the relative orientation of the 15N-13C couplings, and hence the '3C-13C torsion angle. This corresponds to the '~P angle along a peptide backbone when the 13C nuclei are directly-bonded cx- and carbonyl carbons. By recoupling the '5N-13C interactions during the dephasing period, rather than observing their effects during a single rotor cycle at (very) slow spinning speed, we are able to perform the experiment effectively at much higher spinning speeds and with finer time resolution. The result is an experiment that, at least in certain torsion angle regimes, is extremely sensitive to conformation. Because the 13C=O lacks a bound 1H, this represents a unique approach (short of 17O NMR) of applying the 2Q-HLF concept to measuring peptide backbone ~P angles. This provides a general framework for measuring the peptide backbone torsion angle tP in solids that involves 13C-15N dipolar dephasing of 13Ccx-13C o DQ coherence. The specific implementation described here is designed to function effectively at relatively high spinning speeds. The region of high sensitivity to conformation (~P= 1 2O~- 1800) corresponds roughly to the B-sheet structural regime. The extended nature of this type of secondary structure has made it difficult to accurately define the structural details of peptides in which it occurs using distance measuring techniques with outer ranges of 5-6 A. Application of the NCCN 2Q-HLF technique to these systems should be particularly useful in delineating the precise nature of their B-sheet conformation.