Structural Fluctuation of PhotosyntheticAntenna Complexes Studied by Single-Molecule Spectroscopy

蛋白质有许多亚稳定结构。在生理条件下。长期以来，结构波动的重要性一直被指出，因为结构波动在调节蛋白质的功能中起着至关重要的作用。然而，一直没有方法来阐明结构动力学。为了突破，我们的目标是通过对单个蛋白质进行光谱学来获得单个蛋白质的结构信息。由于测量荧光光谱至少需要几毫秒，所以在足够低的温度下进行光观测量，以便跟踪结构动力学。在这项研究中，我们成功地跟踪了一种色素-蛋白质复合物，以了解结构动力学的温度依赖性。实验数据是全球最早的数据，它清楚地表明，结构运动的很大一部分是与温度无关的隧道运动[Oikawa， et. J. Am]。化学。Soc) 130(20008) 48501。单一蛋白质的低温光谱一直局限于近红外区域。该方法已成功应用于光合细菌的光合天线配合物。然而，由于许多酶在可见光区域吸收或发射，因此值得努力延长装置的波长以覆盖可见光区域。为了减少物镜与样品一起浸入液氦中的色差，我们开发了一种反射物镜[Fujiyoshi， et. al. Appl]。理论物理。快报，91(2007)051125。利用反射物镜进行了单颗gfp的双光子激发和荧光光谱测量。光谱是在无背景条件下拍摄的，同一蛋白质的一系列光谱揭示了蛋白质的结构动力学[Fujiyoshi， et. al.]。[j].社会科学与技术，2006(1):1。

A protein has numerous meta-stable structures. In the physiological conditions. The structural fluctuation was pointed out for long years to be important, because the structural fluctuation plays essential role in regulating function of proteins. However, there have been no methods to elucidate the structural dynamics. To break through, we are aiming at getting structural information of single proteins by performing optical spectroscopy of individual proteins. Because measuring a fluorescence spectrum takes at least several milli-seconds, troscopic measurements are performed at temperatures low enough for the structural dynamics to be followed.In this study we have succeeded to follow one pigment-protein complex to know the temperature dependence of structural dynamics. The experimental data, which are the very first data on the globe show clearly that substantial part of the structural movement is temperature-independent tunneling motion [Oikawa, et. al. J. Am. Chem. Soc. 130(20008)48501.].Spectroscopy at low temperature of single proteins has been limited to the near infrared region. The method was successfully applied to photosynthetic antenna complexes of photosynthetic bacteria. Nevertheless it is worth making an effort to extend the wavelength of the setup to cover the visible region, since many enzymes absorb or emit in the visible. In order to reduce the chromatic aberration of the objective, which is immersed into liquid helium together with a sample, we have developed a reflecting objective [Fujiyoshi, et. al. Appl. Phys. Lett. 91 (2007) 051125.]. With the reflecting objective we have made two-photon excitation and measured fluorescence spectrum of single GFPs. The spectra were taken in the background-free condition, and series of spectra of the same protein revealed structural dynamics of the protein [Fujiyoshi, et. al. Phys. Rev. Lett. 100 (2008) 168101.].