Probing chemical shifts of invisible states of proteins with relaxation dispersion NMR spectroscopy:: How well can we do?
Probing chemical shifts of invisible states of proteins with relaxation dispersion NMR spectroscopy:: How well can we do?
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DOI：
10.1021/ja078337p
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发表时间：
2008-02-27
影响因子：
15
通讯作者：
Kay, Lewis E.
中科院分区：
文献类型：
--
作者：
Hansen, D. Flemming;Vallurupalli, Pramodh;Kay, Lewis E.
Carr-Purcell-Meiom-Gill弛豫色散谱已经发展成为研究生物分子低密度、不可见构象的一种强有力的方法。该实验的一个强大特征是可以获得交换构象之间的化学位移差异，提供关于不可见激发态的结构信息。通过开发新的标记方法和核磁共振实验，现在可以测量蛋白质中主干C-13(α)和(CO)-C-13弛豫色散曲线，而不会出现C-13-C-13偶联的并发症。这里给出了这样的测量结果，以及使用N-15和(HN)-H-1原子核探测交换的结果。一个关键的实验设计是选择一个交换系统，在这个系统中，激发态的化学位移是通过独立的测量得知的。因此，可以定量地评估在色散实验中获得的化学位移差的准确性，并确定通常可以获得非常准确的值。实验工作还得到了计算的补充，这些计算表明，在许多情况下，对于汇率和人口落在可以通过弛豫色散量化的范围内的系统，也可以测量出类似的准确移位。提取的化学位移的准确性为获得现在从蛋白质基态记录的化学位移中获得的那种不可见状态的定量结构信息打开了可能性。
Carr-Purcell-Meiboom-Gill relaxation dispersion NMR spectroscopy has evolved into a powerful approach for the study of low populated, invisible conformations of biological molecules. One of the powerful features of the experiment is that chemical shift differences between the exchanging conformers can be obtained, providing structural information about invisible excited states. Through the development of new labeling approaches and NMR experiments it is now possible to measure backbone C-13(alpha) and (CO)-C-13 relaxation dispersion profiles in proteins without complications from C-13-C-13 couplings. Such measurements are presented here, along with those that probe exchange using N-15 and (HN)-H-1 nuclei. A key experimental design has been the choice of an exchanging system where excited-state chemical shifts were known from independent measurement. Thus it is possible to evaluate quantitatively the accuracy of chemical shift-differences obtained in dispersion experiments and to establish that in general very accurate values can be obtained. The experimental work is supplemented by computations that suggest that similarly accurate shifts can be measured in many cases for systems with exchange rates and populations that fall within the range of those that can be quantified by relaxation dispersion. The accuracy of the extracted chemical shifts opens up the possibility of obtaining quantitative structural information of invisible states of the sort that is now available from chemical shifts recorded on ground states of proteins.