Gopalan Anusha B, Yuwen Tairan, Kay Lewis E, Vallurupalli Pramodh
TIFR Centre for Interdisciplinary Sciences, Tata Institute of Fundamental Research Hyderabad, 36/P, Gopanpally Village, Serilingampally Mandal, Ranga Reddy District, Hyderabad, Telangana, 500107, India.
Departments of Molecular Genetics, Biochemistry and Chemistry, University of Toronto, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada.
J Biomol NMR. 2018 Oct;72(1-2):79-91. doi: 10.1007/s10858-018-0208-z. Epub 2018 Oct 1.
Protein conformational changes play crucial roles in enabling function. The Carr-Purcell-Meiboom-Gill (CPMG) experiment forms the basis for studying such dynamics when they involve the interconversion between highly populated and sparsely formed states, the latter having lifetimes ranging from ~ 0.5 to ~ 5 ms. Among the suite of experiments that have been developed are those that exploit methyl group probes by recording methyl H single quantum (Tugarinov and Kay in J Am Chem Soc 129:9514-9521, 2007) and triple quantum (Yuwen et al. in Angew Chem Int Ed Engl 55:11490-11494, 2016) relaxation dispersion profiles. Here we build upon these by developing a third experiment in which methyl H double quantum coherences evolve during a CPMG relaxation element. By fitting single, double, and triple quantum datasets, akin to recording the single quantum dataset at static magnetic fields of B, 2B and 3B, we show that accurate exchange values can be obtained even in cases where exchange rates exceed 10,000 s. The utility of the double quantum experiment is demonstrated with a pair of cavity mutants of T4 lysozyme (T4L) with ground and excited states interchanged and with exchange rates differing by fourfold (~ 900 s and ~ 3600 s), as well as with a fast-folding domain where the unfolded state lifetime is ~ 80 µs.
蛋白质构象变化在实现功能方面起着关键作用。当涉及高丰度态和低丰度态之间的相互转换时，Carr-Purcell-Meiboom-Gill（CPMG）实验构成了研究此类动力学的基础，后者的寿命范围约为0.5至5毫秒。在已开发的一系列实验中，有一些实验通过记录甲基氢单量子（Tugarinov和Kay，《美国化学会志》129：9514 - 9521，2007）和三量子（Yuwen等人，《德国应用化学》国际版55：11490 - 11494，2016）弛豫色散谱来利用甲基基团探针。在此，我们在此基础上开发了第三个实验，其中甲基氢双量子相干在CPMG弛豫元件期间演化。通过拟合单量子、双量子和三量子数据集，类似于在B、2B和3B静磁场下记录单量子数据集，我们表明即使在交换率超过10,000 s的情况下也能获得准确的交换值。通过一对T4溶菌酶（T4L）腔突变体证明了双量子实验的实用性，其基态和激发态互换且交换率相差四倍（约900 s和约3600 s），以及一个快速折叠结构域，其未折叠态寿命约为80微秒。
