Skrynnikov Nikolai R, Dahlquist Frederick W, Kay Lewis E
Protein Engineering Network Center of Excellence, Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
J Am Chem Soc. 2002 Oct 16;124(41):12352-60. doi: 10.1021/ja0207089.
Carr-Purcell-Meiboom-Gill (CPMG) relaxation measurements employing trains of 180 degrees pulses with variable pulse spacing provide valuable information about systems undergoing millisecond-time-scale chemical exchange. Fits of the CPMG relaxation dispersion profiles yield rates of interconversion, relative populations, and absolute values of chemical shift differences between the exchanging states, |Deltaomega|. It is shown that the sign of Deltaomega that is lacking from CPMG dispersion experiments can be obtained from a comparison of chemical shifts in the indirect dimensions in either a pair of HSQC (heteronuclear single quantum coherence) spectra recorded at different magnetic fields or HSQC and HMQC (heteronuclear multiple quantum coherence) spectra obtained at a single field. The methodology is illustrated with an application to a cavity mutant of T4 lysozyme in which a leucine at position 99 has been replaced by an alanine, giving rise to exchange between ground state and excited state conformations with a rate on the order of 1450 s(-1) at 25 degrees C.
采用具有可变脉冲间距的180度脉冲序列进行的 Carr-Purcell-Meiboom-Gill（CPMG）弛豫测量，能为经历毫秒时间尺度化学交换的系统提供有价值的信息。CPMG弛豫色散曲线的拟合可得出互变速率、相对丰度以及交换态之间化学位移差的绝对值|Δω|。研究表明，CPMG色散实验中所缺少的Δω的符号，可通过比较在不同磁场下记录的一对HSQC（异核单量子相干）谱间接维度中的化学位移，或者在单一磁场下获得的HSQC和HMQC（异核多量子相干）谱来得到。该方法通过应用于T4溶菌酶的一个空穴突变体进行了说明，在该突变体中，99位的亮氨酸被丙氨酸取代，导致基态和激发态构象之间以25℃时约1450 s⁻¹的速率进行交换。
