Departments of Molecular Genetics, Biochemistry and Chemistry, The University of Toronto, Toronto, ON, M5S 1A8, Canada.
J Biomol NMR. 2011 Aug;50(4):347-55. doi: 10.1007/s10858-011-9520-6. Epub 2011 Jun 18.
A new pulse sequence is presented for the measurement of relaxation dispersion profiles quantifying millisecond time-scale exchange dynamics of side-chain carbonyl groups in uniformly (13)C labeled proteins. The methodology has been tested using the 87-residue colicin E7 immunity protein, Im7, which is known to fold via a partially structured low populated intermediate that interconverts with the folded, ground state on the millisecond time-scale. Comparison of exchange parameters extracted for this folding 'reaction' using the present methodology with those obtained from more 'traditional' (15)N and backbone carbonyl probes establishes the utility of the approach. The extracted excited state side-chain carbonyl chemical shifts indicate that the Asx/Glx side-chains are predominantly unstructured in the Im7 folding intermediate. However, several crucial salt-bridges that exist in the native structure appear to be already formed in the excited state, either in part or in full. This information, in concert with that obtained from existing backbone and side-chain methyl relaxation dispersion experiments, will ultimately facilitate a detailed description of the structure of the Im7 folding intermediate.
提出了一种新的脉冲序列，用于测量弛豫分散曲线，定量描述侧链羰基在均一（13）C 标记蛋白中毫秒时间尺度交换动力学的毫秒时间尺度交换动力学。该方法已通过 87 残基大肠菌素 E7 免疫蛋白 Im7 进行了测试，已知该蛋白通过部分结构的低 populate 中间体折叠，该中间体在毫秒时间尺度上与折叠的基态相互转换。使用本方法提取的此折叠“反应”的交换参数与从更“传统”（15）N 和骨架羰基探针获得的参数进行比较，证明了该方法的实用性。提取的激发态侧链羰基化学位移表明，Asx/Glx 侧链在 Im7 折叠中间体中主要无结构。然而，在天然结构中存在的几个关键盐桥似乎已经在激发态中部分或全部形成。该信息与现有骨架和侧链甲基弛豫分散实验获得的信息相结合，将最终有助于详细描述 Im7 折叠中间体的结构。
