Yang Yunhuang, Ramelot Theresa A, Ni Shuisong, McCarrick Robert M, Kennedy Michael A
Department of Chemistry and Biochemistry and the Northeast Structural Genomics Consortium, Miami University, Oxford, OH, USA.
Methods Mol Biol. 2014;1091:215-27. doi: 10.1007/978-1-62703-691-7_16.
The success of homodimer structure determination by conventional solution NMR spectroscopy relies greatly on interchain distance restraints (less than 6 Å) derived from nuclear Overhauser effects (NOEs) obtained from (13)C-edited, (12)C-filtered NOESY experiments. However, these experiments may fail when the mixed (13)C-/(12)C-homodimer is never significantly populated due to slow homodimer chain exchange. Thus, knowledge of the homodimer chain exchange kinetics can be put to practical use in preparing samples using the traditional NMR method. Here, we described detailed procedures for using paramagnetic resonance enhancements (PREs) and EPR spectroscopy to measure homodimer chain exchange kinetics. In addition, PRE and EPR methods can be combined to provide mid-range (<30 Å) and long-range (17-80 Å) interchain distance restraints for homodimer structure determination as a supplement to short-range intrachain and interchain distance restraints (less than 6 Å) typically obtained from (1)H-(1)H NOESY experiments. We present a summary of how to measure these distances using NMR-based PREs and EPR-based double electron electron resonance (DEER) measurements and how to include them in homodimer structure calculations.
通过传统的溶液核磁共振光谱法测定同二聚体结构的成功很大程度上依赖于从（13）C编辑、（12）C过滤的NOESY实验获得的核Overhauser效应（NOE）所推导的链间距离限制（小于6 Å）。然而，当由于同二聚体链交换缓慢而使混合的（13）C/（12）C同二聚体从未显著富集时，这些实验可能会失败。因此，同二聚体链交换动力学的知识可实际应用于使用传统核磁共振方法制备样品。在这里，我们描述了使用顺磁共振增强（PRE）和电子顺磁共振光谱法测量同二聚体链交换动力学的详细程序。此外，PRE和EPR方法可以结合起来，为同二聚体结构测定提供中程（<30 Å）和远程（17 - 80 Å）链间距离限制，作为通常从（1）H - （1）H NOESY实验获得的短程链内和链间距离限制（小于6 Å）的补充。我们总结了如何使用基于核磁共振的PRE和基于电子顺磁共振的双电子 - 电子共振（DEER）测量来测量这些距离，以及如何将它们纳入同二聚体结构计算中。
