Korzhnev Dmitry M, Kloiber Karin, Kay Lewis E
Protein Engineering Network Centres of Excellence and the Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
J Am Chem Soc. 2004 Jun 16;126(23):7320-9. doi: 10.1021/ja049968b.
New relaxation dispersion experiments are presented that probe millisecond time-scale dynamical processes in proteins. The experiments measure the relaxation of (1)H-(15)N multiple-quantum coherence as a function of the rate of application of either (1)H or (15)N refocusing pulses during a constant time relaxation interval. In contrast to the dispersion profiles generated from more conventional (15)N((1)H) single-quantum relaxation experiments that depend on changes in (15)N((1)H) chemical shifts between exchanging states, (1)H-(15)N multiple-quantum dispersions are sensitive to changes in the chemical environments of both (1)H and (15)N spins. The resulting multiple-quantum relaxation dispersion profiles can, therefore, be quite different from those generated by single-quantum experiments, so that an analysis of both single- and multiple-quantum profiles together provides a powerful approach for obtaining robust measures of exchange parameters. This is particularly the case in applications to protonated proteins where other methods for studying exchange involving amide proton spins are negatively influenced by contributions from neighboring protons. The methodology is demonstrated on protonated and perdeuterated samples of a G48M mutant of the Fyn SH3 domain that exchanges between folded and unfolded states in solution.
本文介绍了新的弛豫色散实验，该实验用于探测蛋白质中毫秒时间尺度的动力学过程。实验测量了在恒定时间弛豫间隔内，作为¹H或¹⁵N重聚焦脉冲施加速率函数的¹H-¹⁵N多量子相干的弛豫情况。与更传统的¹⁵N(¹H)单量子弛豫实验所产生的色散轮廓不同，后者依赖于交换态之间¹⁵N(¹H)化学位移的变化，¹H-¹⁵N多量子色散对¹H和¹⁵N自旋的化学环境变化均敏感。因此，所得到的多量子弛豫色散轮廓可能与单量子实验所产生的轮廓有很大不同，这样，对单量子和多量子轮廓进行联合分析，为获得可靠的交换参数测量提供了一种强大的方法。在应用于质子化蛋白质时尤其如此，在这种情况下，涉及酰胺质子自旋的其他交换研究方法会受到相邻质子贡献的负面影响。该方法在Fyn SH3结构域的G48M突变体的质子化和全氘代样品上得到了验证，该突变体在溶液中在折叠态和非折叠态之间交换。
