Hu Kaifeng, Vögeli Beat, Clore G Marius
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
J Am Chem Soc. 2007 May 2;129(17):5484-91. doi: 10.1021/ja067981l. Epub 2007 Apr 7.
A carbon-detected TROSY-optimized experiment correlating 1HN, 15N, and 13C' resonances, referred to as c-TROSY-HNCO is presented, in which the 1HN and 15N TROSY effects are maintained in both indirect dimensions, while the directly detected 13C' is doubly TROSY-optimized with respect to 1HN and 15N. A new strategy for sensitivity enhancement, the so-called double echo-antiecho (dEA), is described and implemented in the c-TROSY-HNCO experiment. dEA offers sensitivity enhancement of square root of 2 in both indirect dimensions and is generally applicable to many multidimensional experiments. A carbon-detected HNCO experiment, c-HNCO, without TROSY optimization and sensitivity enhancement is also designed for comparison purposes. Relaxation simulations show that for a protein with a rotational correlation time of 10 ns or larger, the c-TROSY-HNCO experiment displays comparable or higher signal-to-noise (S/N) ratios than the c-HNCO experiment, although the former selects only 1/4 of the initial magnetization relative to the later. The high resolution afforded in the directly detected carbon dimension allows direct measurement of the doublet splitting to extract 1JCalphaC' scalar and 1DCalphaC' residual dipolar couplings. Simulations indicate that the c-TROSY-HNCO experiment offers higher precision (lower uncertainty) compared to the c-HNCO experiment for larger proteins. The experiments are applied to 15N/13C/2H/[Leu,Val]-methyl-protonated IIBMannose, a protein of molecular mass 18.6 kDa with a correlation time of approximately 10 ns at 30 degrees C. The experimental pairwise root-mean-square deviation for the measured 1JCalphaC' couplings obtained from duplicate experiments is 0.77 Hz. By directly measuring the doublet splitting, the experiments described here are expected to be much more tolerant to nonuniform values of 1JCalphaC' (or 1JCalphaC' + 1DCalphaC' for aligned samples) and pulse imperfections due to the smaller number of applied pulses in the "out-and-stay" coherence transfer in the c-HNCO-TROSY experiment relative to conventional 1H-detected "out-and-back" quantitative J correlation experiments. A carbon-detected TROSY-optimized experiment correlating 1HN, 15N, and 13C' resonances, referred to as c-TROSY-HNCO is presented, in which the 1HN and 15N TROSY effects are maintained in both indirect dimensions, while the directly detected 13C' is doubly TROSY-optimized with respect to 1HN and 15N. A new strategy for sensitivity enhancement, the so-called double echo-antiecho (dEA), is described and implemented in the c-TROSY-HNCO experiment. dEA offers sensitivity enhancement of in both indirect dimensions and is generally applicable to many multidimensional experiments.
本文介绍了一种用于关联1HN、15N和13C'共振的碳检测TROSY优化实验，称为c-TROSY-HNCO。在该实验中，1HN和15N的TROSY效应在两个间接维度中均得以保持，而直接检测的13C'相对于1HN和15N进行了双重TROSY优化。本文描述并在c-TROSY-HNCO实验中实施了一种新的灵敏度增强策略，即所谓的双回波-反回波（dEA）。dEA在两个间接维度中都提供了√2的灵敏度增强，并且通常适用于许多多维实验。还设计了一个未进行TROSY优化和灵敏度增强的碳检测HNCO实验，即c-HNCO，用于比较。弛豫模拟表明，对于旋转相关时间为10 ns或更大的蛋白质，c-TROSY-HNCO实验显示出与c-HNCO实验相当或更高的信噪比（S/N），尽管前者相对于后者仅选择了1/4的初始磁化强度。直接检测的碳维度所提供的高分辨率允许直接测量双峰分裂，以提取1JCalphaC'标量和1DCalphaC'残余偶极耦合。模拟表明，对于更大的蛋白质，c-TROSY-HNCO实验比c-HNCO实验具有更高的精度（更低的不确定性）。这些实验应用于15N/13C/2H/[亮氨酸，缬氨酸]-甲基质子化的IIBMannose，这是一种分子量为18.6 kDa的蛋白质，在30℃时相关时间约为10 ns。重复实验测得的1JCalphaC'耦合的实验成对均方根偏差为0.77 Hz。通过直接测量双峰分裂，预计本文所述的实验对1JCalphaC'（或对齐样品中的1JCalphaC'+1DCalphaC'）的不均匀值和脉冲缺陷的耐受性更高，这是因为相对于传统的1H检测“往返”定量J相关实验，c-HNCO-TROSY实验中“出去并停留”相干转移中应用的脉冲数量较少。本文介绍了一种用于关联1HN、15N和13C'共振的碳检测TROSY优化实验，称为c-TROSY-HNCO。在该实验中，1HN和15N的TROSY效应在两个间接维度中均得以保持，而直接检测的13C'相对于1HN和15N进行了双重TROSY优化。本文描述并在c-TROSY-HNCO实验中实施了一种新的灵敏度增强策略，即所谓的双回波-反回波（dEA）。dEA在两个间接维度中都提供了灵敏度增强，并且通常适用于许多多维实验。
