Department of Molecular Genetics, The University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
J Am Chem Soc. 2010 Aug 18;132(32):10992-5. doi: 10.1021/ja104578n.
A pulse scheme is presented for quantifying millisecond time scale chemical exchange processes in proteins by measuring (1)H CPMG relaxation dispersion profiles of (13)CHD(2) methyl groups. The use of (13)CHD(2) isotopomers for (1)H methyl dispersion experiments eliminates problems with interconversion between differentially relaxing proton transitions that complicate the extraction of accurate exchange parameters when (13)CH(3) probes are used. Good agreement is demonstrated between extracted chemical shift differences from fits of dispersion profiles and the corresponding differences measured independently on a model exchanging system, validating the experiment. The methodology is applied to the gating residues of the T. acidiphilium proteasome that are shown to undergo extensive motion on the millisecond time scale.
本文提出了一种通过测量 (13)CHD2 甲基的 (1)H CPMG 弛豫分散曲线来量化蛋白质中毫秒时间尺度化学交换过程的脉冲方案。使用 (13)CHD2 异构体进行 (1)H 甲基分散实验消除了在使用 (13)CH3 探针时，不同弛豫质子跃迁之间的互变所带来的问题，这种互变会使准确提取交换参数变得复杂。从分散曲线拟合中提取的化学位移差异与在模型交换体系中独立测量的相应差异之间具有良好的一致性，验证了该实验。该方法应用于 T. acidiphilium 蛋白酶的门控残基，结果表明这些残基在毫秒时间尺度上发生了广泛的运动。
