Photodynamics in Second Generation Fluorescent Proteins

从一种相对鲜为人知的水母中分离出的天然荧光蛋白(FP)可以被克隆并在其他生物中表达的发现，导致了生物成像方面的革命性进步。绿色荧光蛋白的基因与目的蛋白的基因拼接确保了无论何时表达目标蛋白，它都不可逆转地与其荧光伙伴结合。通过使用标准的荧光显微镜工具，细胞生物学家可以在活细胞中观察蛋白质产生、执行其功能并最终被降解的过程。在这一初步发现后不久，几个FP的突变体被创造出来，这些突变体改变了光谱，允许对多种物种进行成像，并通过荧光共振能量转移，研究蛋白质与蛋白质的相互作用。几年前，FP家族的另一个分支被从珊瑚礁中分离出来。关于这些新的FP，最令人兴奋的发现是它们是光活性的--也就是说，它们的光学性质可以通过特定波长的光照射来操纵。例如，绿色发射蛋白质可以通过紫外线辐射转化为红色发射体。这导致了“光学高亮”，即在空间的特定点产生不同颜色的蛋白质，随后该群体的进化可以与细胞中所有其他相同的蛋白质分开研究。也许更重要的是发现了可光激活的蛋白质，这种蛋白质在受到照射之前是非荧光的(不同波长的光发生相反的过程)。这使得在任何时候只有几种蛋白质发出荧光成为可能。因此，极高对比度的单分子成像成为可能，使蛋白质运动的超(纳米级)分辨率研究成为可能。这被称为第二次计划生育革命。我们的目标是理解我们所称的第二代FP的光活性行为背后的光物理。这一多学科计划得到了当地和国际合作的支持。解开第二代FP的激发态化学的主要工具将是荧光，特别是超快时间分辨荧光，其中我们记录了发射强度和光谱的时间行为，分辨率低于50飞秒。这为研究激发态势能面上的分子动力学提供了独特的见解。这些动力学将作为氢化反应的函数来研究，以揭示质子转移在光活化中的作用，通过诱变来研究蛋白质基质的作用，以pH来探索不同残基和温度对滴定的影响，以寻找光活化中激发态能垒的存在和高度。这些数据将根据合作者实验室正在进行的结构研究进行进一步解释。除了这些对天然蛋白质及其突变体的研究外，我们还将把我们的研究扩展到含有非天然氨基酸的FP，这将使我们能够更好地控制蛋白质的光物理性质。对完整蛋白质的这些研究将由实验室合成的发色团单位的研究来补充。对裸发色团的光物理控制因素的详细研究将为解释蛋白质动力学提供重要的基础数据，也将为测试生色团激发态势能表面的理论计算提供重要的基础数据。这项研究是必要的，因为需要更好的设计和更具体的FP，不仅作为活细胞成像的探针，而且作为光活性传感器分子，这将使FP能够用于绘制环境的位置和化学性质。这一目标的成功将极大地扩大FP在生命科学中的应用范围，并导致第三次FP革命。

The discovery that a naturally fluorescent protein (FP) isolated from a relatively obscure jellyfish could be cloned and expressed in other organisms led to revolutionary advances in bioimaging. The splicing of the gene for the green fluorescent protein to one for a protein of interest ensures than whenever the target protein is expressed it is irreversibly bound to its fluorescent partner. By using the standard tools of fluorescence microscopy the cell biologist can then observe the protein in a living cell as it is created, performs its function and is ultimately degraded. Soon after this initial discovery several mutants of FPs were created which modified the spectrum, allowing imaging of multiple species, and, through fluorescence resonance energy transfer, the study of protein-protein interactions. A few years ago another branch of the FP family was isolated from reef corals. The most exciting finding concerning these new FPs is that they are photoactive - that is their optical properties can be manipulated by irradiation with light of a specific wavelength. For example a green emitting protein can be converted to a red emitter through UV irradiation. This leads to 'optical highlighting' in which differently coloured proteins are generated at specific points in space and the subsequent evolution of that population can be studied separately from all the otherwise identical proteins in the cell. Perhaps even more significant has been the discovery of photoactivateable proteins, which are non fluorescent until irradiated (with the reverse process occurring for different wavelength light). This makes it possible to make only a few proteins fluorescent at any one time. As a result extremely high contrast single molecule imaging becomes possible, permitting super (nanometre scale) resolution studies of protein motion. This has been referred to as the second FP revolution. Our objective is to understand the photophysics underlying the photoactive behaviour in what we term second generation FPs.This multidisciplinary programme is supported by local and international collaborations.The main tool for unraveling the excited state chemistry of second generation FPs will be fluorescence, particularly ultrafast time resolved fluorescence, in which we record the temporal behaviour of the emission intensity and spectrum with sub 50 femtosecond resolution. This affords unique insights into molecular dynamics on the excited state potential energy surface. These dynamics will be studied as a function of deuteration to unravel the role of proton transfer in the photoactivation, mutagenesis to investigate the role of the protein matrix, pH to probe the effect of titration of different residues and temperature to look for the existence and height of excited state energy barriers to photoactivation. The data will be further interpreted in the light of structural studies underway in collaborators laboratories. In addition to these studies of natural proteins and their mutants we will extend our investigations to FPs containing unnatural amino acids, which will permit finer control of the photophysical properties of the protein. These studies of intact proteins will be complemented by investigations of the chromophore unit synthesised in the laboratory. A detailed study of the factors controlling the photophysics of the bare chromophore will provide vital underpinning data for interpreting protein dynamics, and also for testing theoretical calculations of chromophore excited state potential energy surfaces.This study is essential because of the need for better designed and more specific FPs, to act not only as probes for live cell imaging, but also as photoactive sensor molecules, which will allow FPs to be used to map out both the location and the chemical nature of the environment. The success of this objective will be to dramatically widen the range of applications of FPs in life sciences, and lead to a third FP revolution.