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Time-Resolved Vibrational Spectroscopic Investigation of Ultrafast Protein Dynamics Coupled with Photoreaction

超快蛋白质动力学与光反应耦合的时间分辨振动光谱研究

基本信息

批准号：
12045264
负责人：
KITAGAWA Teizo
金额：
$ 9.09万
依托单位：
Center for Integrative Bioscience, Okazaki National Research Institutes
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

In many biological systems a localized small structural change extends spatially to mesoscopic dimensions to achieve a physiological function, and higher-order structural changes of proteins are essential to this process. Myoglobin (Mb) is one of the best molecules for studying such features of proteins, because photodissociation of CO from carbonmonoxyMb (MbCO), a localized reaction, takes place within 50 fs like a step-function with a quantum yield of nearly unity. It is know from x-ray studies that the iron atom moves out of the porphyrin plane by 〜0.3 A and the core-size expands by 0.05A upon deligation of CO. To explore the protein dynamics involved in this process, we have investigated picosecond time-resolved resonance Raman spectra of photodissociated MbCO.Close inspection of the spectra of deoxyMb following CO photolysis in the 1300-1650 cm^<-1> region revealed that the core-size expansion of porphyrin ring is completed within the instrumental response time (〜2 ps). In contrast, changes in the intensity and frequency of the Fe-His stretching mode (VFe-His) occurred in picoseconds, suggesting appreciable time evolution for the Fe displacement from the porphyrin plane. The same behaviors were observed for the model compound of the heme group without protein matrix. Therefore, this reflects an intrinsic property of heme itself. On the other hand, the frequency of the VFe-His mode changed with a time constant of 〜100 ps. This frequency change was not seen for the model compound without the protein matrix. Therefore, this must be caused by tertiary structural changes of the protein. Temporal changes of the anti-Stokes Raman intensity of the V_4 and V_7 bands demonstrated immediate generation of the vibrationally excited heme upon photolysis and subsequent decay of the vibrationally excited population with the time constants of 1.1±0.6 and 1.9±0.6 ps, respectively.

在许多生物系统中，局部的小结构变化在空间上扩展到介观维度以实现生理功能，而蛋白质的高阶结构变化对这一过程至关重要。肌红蛋白（Mb）是研究蛋白质这类特征的最佳分子之一，因为一氧化碳与一氧化碳（MbCO）的光解是一个局部反应，像阶跃函数一样在50秒内发生，量子产率几乎是一致的。从x射线研究中可以得知，在CO的作用下，铁原子向卟啉平面外移动了~ 0.3 A，核尺寸扩大了0.05A。为了探索这一过程中涉及的蛋白质动力学，我们研究了光解MbCO的皮秒时间分辨共振拉曼光谱。在1300 ~ 1650 cm^<-1>范围内对CO光解后脱氧ymb的光谱进行了仔细观察，结果表明卟啉环在仪器响应时间（~ 2ps）内完成了核心尺寸的扩展。相比之下，Fe- his拉伸模式（VFe-His）的强度和频率的变化发生在皮秒内，表明Fe从卟啉平面位移的时间演变相当可观。不加蛋白基质的血红素组模型化合物表现出相同的行为。因此，这反映了血红素本身的内在特性。另一方面，VFe-His模式的频率以~ 100 ps的时间常数变化，这种频率变化在没有蛋白质基质的模型化合物中没有看到。因此，这一定是由蛋白质的三级结构变化引起的。V_4和V_7的抗stokes拉曼强度随时间的变化表明，在光解作用下，振动激发的血红素立即产生，随后振动激发的种群衰变，时间常数分别为1.1±0.6和1.9±0.6 ps。