STRUCT DETERMINATION OF UNIFORMLY LABELED MOLECULES BY SSNMR DIPOLAR RECOUPLING

我们提出了各种结构约束，从 均匀的“C，”N标记小分子中的偶极重新耦合， 包括促樱桃多肽甲酰-蛋氨酸-亮氨酸-苯丙氨酸和 大环内酯类抗生素红霉素A对HCCH和HNCH的延展作用 扭转角法利用新开发的T-MREV序列 (Hohwy和同事)，并使用两个化学位移维度来 同时提供多个投影角度的约束。为 最佳效率，执行同核偏振转移 利用一类y编码的双量子序列(C7及其 补偿模拟物)，以及绝热异核转移 交叉极化。我们的方法与以前的方法进行了比较， 例如依赖于多个IC脉冲H-X重新耦合或 双量子相干的局域场演化。在前者中 在这种情况下，从n个脉冲引入大量伪影；在 后者，约束仅限于直接绑定的邻居。 衡量多重债券的方法提供了实质性的优势 而不是单键测量来确定结构，因为 独立扭转角的冗余约束 ‘HXX2H投影角度减少了误差的传播。 CH2和CH3组的并发症，松弛，和众多较弱 耦合用高效的多自旋数值模拟 模拟。除了基于HX的约束之外，我们还演示了 15N 13C 13C 15N 14测量的应用，以及所需的 双量子13C_(13)C的化学位移分辨 尺寸。对于统一标记系统中的距离测量，我们 探索两种可能性。对于异核距离， 对REDOR实验和选择性CP方法进行了比较。为 同核自旋对，我们考虑自旋对的程度 具有天然优势的化学位移差异可以 被利用来提取残基内和残基间的距离。例如, 旋转共振抖动(R2T)用于重新耦合CO-CH3对 并进行了选择性实验室和旋转框架再耦合实验。 用于CO-CO或CH3-CH3对。在所有讨论的实验中， 极化转移效率极佳(&gt；50%)和内部 控制是存在的。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 是存在的。

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.