Ball Graeme, Meenan Nicola, Bromek Krystyna, Smith Brian O, Bella Juraj, Uhrín Dusan
School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK.
J Magn Reson. 2006 May;180(1):127-36. doi: 10.1016/j.jmr.2006.01.017. Epub 2006 Feb 21.
We have developed new 2D and 3D experiments for the measurement of C(alpha)-H(alpha) residual dipolar coupling constants in (13)C and (15)N labelled proteins. Two experiments, 2D (HNCO)-(J-CA)NH and 3D (HN)CO-(J-CA)NH, sample the C(alpha)-H(alpha) splitting by means of C(alpha) magnetization, while 2D (J-HACACO)NH and 3D J-HA(CACO)NH use H(alpha) magnetization to achieve a similar result. In the 2D experiments the coupling evolution is superimposed on the evolution of the (15)N chemical shifts and the IPAP principle is used to obtain (1)H-(15)N HSQC-like spectra from which the splitting is determined. The use of a third dimension in 3D experiments reduces spectral overlap to the point where use of an IPAP scheme may not be necessary. The length of the sampling interval in the J-dimension of these experiments is dictated solely by the relaxation properties of C(alpha) or H(alpha) nuclei. This was made possible by the use of C(alpha) selective pulses in combination with either a DPFGSE or modified BIRD pulses. Inclusion of these pulse sequence elements in the J-evolution periods removes unwanted spin-spin interactions. This allows prolonged sampling periods ( approximately 25 ms) yielding higher precision C(alpha)-H(alpha) splitting determination than is achievable with existing frequency based methods.
我们已经开发出用于测量(^{13}C)和(^{15}N)标记蛋白质中(Cα-Hα)剩余偶极耦合常数的新二维和三维实验。两个实验，二维((HNCO)-(J - CA)NH)和三维((HN)CO-(J - CA)NH)，通过(Cα)磁化来采样(Cα-Hα)分裂，而二维((J - HACACO)NH)和三维(J - HA(CACO)NH)使用(Hα)磁化来获得类似结果。在二维实验中，耦合演化叠加在(^{15}N)化学位移的演化上，并且使用IPAP原理来获得(^{1}H - ^{15}N)类似HSQC的谱图，从中确定分裂。三维实验中使用第三维将光谱重叠减少到可能不需要使用IPAP方案的程度。这些实验在(J)维度上的采样间隔长度仅由(Cα)或(Hα)核的弛豫特性决定。这通过使用(Cα)选择性脉冲与DPFGSE或改进的BIRD脉冲相结合得以实现。在(J)演化期包含这些脉冲序列元素可消除不需要的自旋 - 自旋相互作用。这允许延长采样周期（约25毫秒），从而比现有基于频率的方法能够更精确地确定(Cα-Hα)分裂。
