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Development of Highly Sensitive Infrared Spectrometer with Femtosecond Time Resolution

飞秒时间分辨率高灵敏红外光谱仪的研制

基本信息

批准号：
11554028
负责人：
YOSHIHARA Keitaro
金额：
$ 6.85万
依托单位：
JAPAN ADVANCED INSTITUTE OF SCIENCE AND TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

The design and performance of a femtosecond time-resolved mid-infrared transient absorption spectrometer is illustrated. The spectrometer was developed from a set of commercially available Ti : sapphire femtosecond lasers utilizing difference frequency mixing technique. With this spectrometer, the transient absorption in the mid-infrared region from 800 to 2800 cm^<-1> was detected with a sensitivity of 1×10^<-4> for difference absorbance at a repetition rate of 1 kHz. With a sub-picosecond visible or ultraviolet pump, a time-resolution of 250fs has been obtained. A study on the binding and dissociation processes of carbon monoxide in myoglobin is presented as an example of its applications.CooA, which is a transcriptional regulator heme protein allosterially triggered by CO, is studied by femtosecond visible-pump mid-IR-probe spectroscopy. Transient bleaching upon excitation of the heme into Soret band is detected at around 1979cm^<-1>, which is the absorption region of the CO bound to the heme. The bleach signal shows a nonexponential decay with time constants of 56 ps and 290 ps, caused by the rebinding of the CO to the heme. About 97% of dissociated CO recombines geminately. The geminate recombination rate in CooA is significantly faster than that in myoglobin and hemoglobin. The angle of the bound CO with respect to the porphyrin plane is calculated to be about 78 degrees based on the anisotropy measurements. A shift of the bleach mid-IR spectrum of the bound CO is detected having characteristic time of 160 ps. It is suggested that the spectral shift is caused by a difference in the frequency of the bound CO in different protein conformations, particularly in active conformation and in intermediate one. Thus, the biologically relevant conformation change in CooA was traced. Possible assignment of the observed conformation change is discussed.

介绍了飞秒时间分辨中红外瞬态吸收光谱仪的设计和性能。该光谱仪是由一套市售的钛宝石飞秒激光器，利用差频混合技术。利用该光谱仪，在800至2800 cm-1的中红外区域中的瞬态吸收<-1>被检测到，<-4>对于1 kHz的重复频率下的差异吸光度的灵敏度为1×10 - 1。在亚皮秒可见光或紫外泵浦下，获得了250 fs的时间分辨率。以CO在肌红蛋白中的结合和解离过程为例，采用飞秒可见泵浦中红外探针光谱技术研究了CO诱导的血红素蛋白CooA的变构反应。在1979 cm-1附近检测到血红素激发到Soret带时的瞬时漂白<-1>，这是与血红素结合的CO的吸收区域。漂白信号表现出非指数衰减的时间常数为56 ps和290 ps，所造成的CO的重新绑定的血红素。约97%的解离CO以成对方式重组。CooA的成对重组速率明显快于肌红蛋白和血红蛋白。结合CO相对于卟啉平面的角度计算为约78度的各向异性测量的基础上。检测到结合CO的漂白中红外光谱的位移，其特征时间为160 ps。有人建议，光谱移动是由不同的蛋白质构象，特别是在活性构象和中间的结合CO的频率的差异。因此，追踪了CooA中的生物学相关构象变化。可能的分配所观察到的构象变化进行了讨论。