A "just-in-time' HN(CA)CO experiment for the backbone assignment of large proteins with high sensitivity
A "just-in-time' HN(CA)CO experiment for the backbone assignment of large proteins with high sensitivity
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DOI：
10.1016/j.jmr.2006.04.001
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发表时间：
2006-07-01
影响因子：
2.2
通讯作者：
Zhou, Pei
中科院分区：
文献类型：
--
作者：
Werner-Allen, Jon W.;Jiang, Ling;Zhou, Pei
在常用的骨干实验中，由于快速(CO)-C-13横向弛豫和被动C-13(α)-C-13（β）耦合，HNCACO存在无法解决的灵敏度限制。在这里，我们提出了一个高灵敏度的“即时”（JIT） HN(CA)CO脉冲序列，该脉冲序列在实时收集(CO)-C-13位移的同时，均匀地重新聚焦C-13(α)-C-13（β）耦合。采用H-2/C-13/N-15标记的29 kDa HCA II蛋白样品，在15℃条件下，对3-D JIT HN(CA)CO、ct - hmqc为基础的对照和hsqc为基础的选择性C-13（α）反转脉冲的对照进行了灵敏度比较。JIT实验显示，与ct - hmqc为基础的实验相比，信号增强了42%。与基于hsqc的实验相比，JIT实验对C-13（α）再聚焦和C-13(α)-C-13（β）解耦的残基敏感度降低了16%。然而，对于剩余的残留物，JIT光谱显示比基于hsqc的实验平均灵敏度增益106%。高灵敏度JIT HNCACO实验尤其有利于研究大蛋白，无论残基类型如何，都能提供(CO)-C-13共振信息。(c) 2006爱思唯尔公司版权所有。
Among the suite of commonly used backbone experiments, HNCACO presents an unresolved sensitivity limitation due to fast (CO)-C-13 transverse relaxation and passive C-13(alpha)-C-13(beta) coupling. Here, we present a high-sensitivity 'just-in-time' (JIT) HN(CA)CO pulse sequence that uniformly refocuses C-13(alpha)-C-13(beta) coupling while collecting (CO)-C-13 shifts in real time. Sensitivity comparisons of the 3-D JIT HN(CA)CO, a CT-HMQC-based control, and a HSQC-based control with selective C-13(alpha) inversion pulses were performed using a H-2/C-13/N-15 labeled sample of the 29 kDa HCA II protein at 15 degrees C. The JIT experiment shows a 42% signal enhancement over the CT-HMQC-based experiment. Compared to the HSQC-based experiment, the JIT experiment is 16% less sensitive for residues experiencing proper C-13(alpha) refocusing and C-13(alpha)-C-13(beta) decoupling. However, for the remaining residues, the JIT spectrum shows a 106% average sensitivity gain over the HSQC-based experiment. The high-sensitivity JIT HNCACO experiment should be particularly beneficial for studies of large proteins to provide (CO)-C-13 resonance information regardless of residue type. (c) 2006 Elsevier Inc. All rights reserved.