Hansen D Flemming, Vallurupalli Pramodh, Kay Lewis E
Department of Medical Genetics, The University of Toronto, Toronto, Ontario, Canada, M5S 1A8.
J Phys Chem B. 2008 May 15;112(19):5898-904. doi: 10.1021/jp074793o. Epub 2007 Nov 15.
A new (15)N constant-time relaxation dispersion pulse scheme for the quantification of millisecond time-scale exchange dynamics in proteins is presented. The experiment differs from previously developed sequences in that it includes (1)H continuous-wave decoupling during the (15)N Carr-Purcell-Meiboom-Gill (CPMG) pulse train that significantly improves the relaxation properties of (15)N magnetization, leading to sensitivity gains in experiments. Moreover, it is shown that inclusion of an additional (15)N 180 degrees refocusing pulse (phase cycled +/- x) in the center of the CPMG pulse train, consisting of 1(5)N 180 degrees (y) pulses, provides compensation for pulse imperfections beyond the normal CPMG scheme. Relative to existing relaxation-compensated constant-time relaxation dispersion pulse schemes, nu(CPMG) values that are only half as large can be employed, offering increased sensitivity to slow time-scale exchange processes. The robustness of the methodology is illustrated with applications involving a pair of proteins: an SH3 domain that does not show millisecond time-scale exchange and an FF domain with significant chemical exchange contributions.
提出了一种用于定量蛋白质中毫秒时间尺度交换动力学的新型（15）N 恒时弛豫色散脉冲方案。该实验与先前开发的序列不同之处在于，它在（15）N Carr-Purcell-Meiboom-Gill（CPMG）脉冲序列期间包含（1）H 连续波去耦，这显著改善了（15）N 磁化的弛豫特性，从而提高了实验的灵敏度。此外，研究表明，在由 1（5）N 180°（y）脉冲组成的 CPMG 脉冲序列中心加入一个额外的（15）N 180°重聚焦脉冲（相位循环为±x），可补偿常规 CPMG 方案之外的脉冲缺陷。相对于现有的弛豫补偿恒时弛豫色散脉冲方案，可以采用仅为其一半大小的 ν（CPMG）值，从而提高对慢时间尺度交换过程的灵敏度。通过涉及一对蛋白质的应用说明了该方法的稳健性：一个不显示毫秒时间尺度交换的 SH3 结构域和一个具有显著化学交换贡献的 FF 结构域。
