A Modified Infrared Spectrometer with High Time Resolution and Its Application for Investigating Fast Conformational Changes of the GTPase Ras
A Modified Infrared Spectrometer with High Time Resolution and Its Application for Investigating Fast Conformational Changes of the GTPase Ras
复制标题
改进的高时间分辨率红外光谱仪及其在研究 GTPase Ras 快速构象变化中的应用
作者：
Jie Lin;K. Gerwert;C. Kötting
时间分辨红外光谱是研究蛋白质和蛋白质相互作用的重要工具。许多生物过程的研究是可能的，通过笼状化合物，释放生物活性物质的光激活。一些笼状化合物可以提供亚纳秒时间分辨率，例如，对羟基苯甲酰甲基-鸟苷5 ′-三磷酸（GTP）在皮秒内形成GTP。然而，快速扫描傅里叶变换红外（FT-IR）光谱仪在单次拍摄实验的时间分辨率是有限的，约10毫秒。在这里，我们使用红外二极管激光器，而不是传统的globar和实现100纳秒的时间分辨率。这允许在室温下快速Rasoff到Rason构象变化的时间分辨测量。我们定量了该反应的活化参数，发现该反应的活化自由能主要是双稳态的。在效应Raf（RafRBD）的Ras结合结构域的存在下的相同反应的调查揭示了四个数量级的更快的反应，表明RasRafRBD复合物的形成直接诱导构象变化。宽调谐量子级联激光器的最新发展将进一步提高时间分辨率和可用性的设置。报道的100 ns时间分辨率是迄今为止非重复性实验中最好的。
Time-resolved infrared spectroscopy is a valuable tool for the investigation of proteins and protein interactions. The investigation of many biological processes is possible by means of caged compounds, which set free biologically active substances upon light activation. Some caged compounds could provide subnanosecond time resolution, e.g., para-hydroxyphenacyl-guanosine 5'-triphosphate (GTP) forms GTP in picoseconds. However, the time resolution in single shot experiments with rapid-scan Fourier transform infrared (FT-IR) spectrometers is limited to about 10 ms. Here we use an infrared diode laser instead of the conventional globar and achieve a time resolution of 100 ns. This allows for the time-resolved measurement of the fast Rasoff to Rason conformational change at room temperature. We quantified the activation parameters for this reaction and found that the free energy of activation for this reaction is mainly enthalpic. Investigation of the same reaction in the presence of the Ras binding domain of the effector Raf (RafRBD) reveals a four orders of magnitude faster reaction, indicating that RasRafRBD complex formation directly induces the conformational change. Recent developments of broadly tunable quantum cascade lasers will further improve time resolution and usability of the setup. The reported 100 ns time resolution is the best achieved for a non-repetitive experiment so far.
DOI：
10.1073/pnas.0712095105
发表时间：
2008-04-29
影响因子：
11.1
作者：
Koetting, Carsten;Kallenbach, Angela;Gerwert, Klaus
通讯作者：
Gerwert, Klaus