STRUCT DETERMINATION OF UNIFORMLY LABELED MOLECULES BY SSNMR DIPOLAR RECOUPLING

我们提出了各种结构性的限制， 偶极再偶联在均匀-“C“，N-标记的小分子， 包括趋化肽甲酰基-Met-Leu-Phe和 大环内酯类抗生素红霉素A HCCH和HNCH的扩展 扭转角方法利用新开发的T-MREV序列 （Hohwy和同事），并采用两个化学位移尺寸， 提供多个投影角度的同时约束。 为 最佳效率，进行同向偏振转换 利用y-编码的双量子序列类（C7及其 补偿类似物），和异频传输的绝热 交叉极化 我们的方法与以前的方法相比， 例如那些依赖于多个IC脉冲H-X再耦合或 局域场演化对双量子相干性的影响。 在前 在这种情况下，从n个脉冲引入了大量伪影; 后者的约束限于直接绑定的邻居。 对多重键的测量提供了实质性的优势 与用于结构测定的单键测量相比， 单独扭转角的冗余约束 HXX 2 H投影角减少了误差的传播。 的 CH 2和CH 3组的并发症，松弛，以及许多较弱的 耦合建模与高效的多自旋数值 模拟 除了'HX为基础的约束，我们证明 应用15 N 13 C 13 C 15 N 14测量， 双量子~（13）C_（13）C化学位移的分辨 维度 对于均匀标记系统中的距离测量，我们 探索两种可能性。 对于异质距离， 比较了REDOR实验和选择性CP方法。 为 我们考虑自旋对在多大程度上 具有自然有利的化学位移差， 用于提取残基内和残基间距离。 比如说， 旋转共振挠痒痒（R2 T）用于重新偶联CO-CH 3对 和选择性的实验室和旋转框架重新耦合实验， 用于CO-CO或CH 3-CH 3对。 在所讨论的所有实验中， 偏振转换效率优异（>50%）， 控件存在。 因此，这些方法可以扩展到 更大的生物分子，假设有足够的分辨率和灵敏度 存在.

We present a variety of structural constraints derived from dipolar recoupling in uniformly- "C, "N-labeled small molecules, including the chernotactic peptide formyl-Met-Leu-Phe and the macrolide antibiotic erythromycin A. Extensions of HCCH and HNCH torsion angle methods exploit the newly developed T-MREV sequence (Hohwy and co-workers) and employ two chemical shift dimensions to provide simultaneous constraints of several projection angles. For optimal efficiency, homonuclear polarization transfer is performed with the class of y-encoded double-quantum sequences (C7 and its compensated analogs), and heteronuclear transfer by adiabatic cross-polarization. Our approach is compared with previous methods, such as those that rely upon multiple ic pulse 'H-X recoupling or local field evolution on the double-quantum coherence. In the former case, substantial artifacts are introduced from the n pulses; in the latter, constraints are limited to directly bonded neighbors. Measuremen ts over multiple bonds provide substantial advantages versus single bond measurements for structure determination, because redundant constraints of individual torsion angles from separate 'HXX2H projection angles reduce propagation of error. The complications of CH2 and CH3 groups, relaxation, and numerous weaker couplings are modeled with highly efficient multi-spin numerical simulations. In addition to the 'HX based constraints, we demonstrate application of the 15N 13C 13C 15N 14 measurement, with the requisite resolution of chemical shifts in the double quantum 13C_13 C dimension. For distance measurements in uniformly labeled systems, we explore two possibilities. For heteronuclear distances, variations of the REDOR experiment and sele * ctive CP approaches are compared. For homonuclear spin pairs, we consider the extent to which spin pairs with naturally advantageous chemical shift differences can be exploited to extract intra- and inter-residue distances. For example, rotational resonance tickling (R2T) is used to recouple CO-CH3 pairs and selective laboratory and rotating frame recoupling experiments are used for CO-CO or CH3-CH3pairs. In all experiments discussed, the efficiency of polarization transfer is excellent (>50%) and internal controls are present. Therefore, these methods may be extended to larger biomolecules, assuming sufficient resolution and sensitivity exist.