Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-520, United States.
J Phys Chem Lett. 2020 Jul 16;11(14):5643-5648. doi: 10.1021/acs.jpclett.0c01636. Epub 2020 Jul 1.
An approach for the quantitative description of the kinetics of very fast exchange processes (τ < 50-100 μs) associated with transient, reversible protein oligomerization, is presented. We show that on-resonance N- measurements conducted as a function of protein concentration at several spin-lock radio frequency field strengths are indispensable for unambiguous determination of the rate constants for interconversion between monomeric and higher order oligomeric species. The approach is experimentally demonstrated on the study of fast, reversible tetramerization of the full-length Huntingtin exon 1 protein, htt, responsible for Huntington's disease. Incorporation of concentration-dependent N- data, obtained from on-resonance measurements performed at three spin-lock field strengths, into analysis of the kinetic scheme describing reversible tetramerization of htt allowed us to uniquely determine the rate constants of interconversion between the various species. This approach serves as a valuable complement to the existing array of NMR techniques for studying early, transient oligomerization events in protein aggregation pathways.
本文提出了一种用于定量描述与瞬态、可逆蛋白质寡聚化相关的非常快速交换过程（τ < 50-100 μs）的动力学的方法。我们表明，在几个自旋锁定射频场强度下作为蛋白质浓度函数进行的共振 N-测量对于明确确定单体和更高阶寡聚体之间的转化速率常数是必不可少的。该方法在研究全长 Huntingtin 外显子 1 蛋白 htt（导致亨廷顿病）的快速、可逆四聚化的研究中得到了实验验证。将在三个自旋锁定场强度下进行的共振测量获得的浓度依赖性 N-数据纳入描述 htt 可逆四聚化的动力学方案的分析中，使我们能够唯一地确定各种物种之间的转化速率常数。该方法为研究蛋白质聚集途径中早期瞬态寡聚化事件的现有一系列 NMR 技术提供了有价值的补充。
