Multiple-site exchange in proteins studied with a suite of six NMR relaxation dispersion experiments: An application to the folding of a Fyn SH3 domain mutant
Multiple-site exchange in proteins studied with a suite of six NMR relaxation dispersion experiments: An application to the folding of a Fyn SH3 domain mutant
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DOI：
10.1021/ja054550e
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发表时间：
2005-11-09
影响因子：
15
通讯作者：
Kay, LE
中科院分区：
文献类型：
--
作者：
Korzhnev, DM;Neudecker, P;Kay, LE
Fyn SH 3结构域的N-15标记的、高氘代Gly 48 Met突变体的三位点交换折叠反应已经在25 ℃下使用一套六个CPMG型弛豫分散实验来表征，该实验测量交换对蛋白质中骨架H-1和N-15横向弛豫速率的贡献。它示出，这套实验允许提取的所有参数的多位点交换过程中的一个强大的方式，包括交换状态之间的化学位移差异，从一个数据集记录在只有一个单一的温度。适合的交换折叠、中间和未折叠状态的群体分别为94%、0.7%和5%。尽管中间体的一小部分，结构信息获得这种状态是一致的图片SH 3结构域折叠，已经出现在其他研究。两者合计，六个分散实验有利于完整重建的H-1-N-15相关光谱的展开和中间状态，即使是最敏感的NMR实验中是“不可见的”。
The three-site exchange folding reaction of an N-15-labeled, highly deuterated Gly48Met mutant of the Fyn SH3 domain has been characterized at 25 degrees C using a suite of six CPMG-type relaxation dispersion experiments that measure exchange contributions to backbone H-1 and N-15 transverse relaxation rates in proteins. It is shown that this suite of experiments allows the extraction of all the parameters of this multisite exchange process in a robust manner, including chemical shift differences between exchanging states, from a data set recorded at only a single temperature. The populations of the exchanging folded, intermediate, and unfolded states that are fit are 94, 0.7, and 5%, respectively. Despite the small fraction of the intermediate, structural information is obtained for this state that is consistent with the picture of SH3 domain folding that has emerged from other studies. Taken together, the six dispersion experiments facilitate the complete reconstruction of H-1-N-15 correlation spectra for the unfolded and intermediate states that are "invisible" in even the most sensitive of NMR experiments.