Photodynamics in Second Generation Fluorescent Proteins

从一种相对不知名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体中表达，这一发现导致了生物成像领域的革命性进步。绿色荧光蛋白的基因与目标蛋白的基因的剪接确保了无论何时目标蛋白被表达，它都不可逆地与它的荧光伙伴结合。通过使用荧光显微镜的标准工具，细胞生物学家可以观察活细胞中的蛋白质，因为它是产生的，执行其功能和最终降解。在这个最初的发现之后不久，几个FPs突变体被创造出来，它们修改了光谱，允许对多个物种进行成像，并通过荧光共振能量转移，研究蛋白质之间的相互作用。几年前，FP家族的另一个分支从珊瑚礁珊瑚中分离出来。关于这些新型FPs，最令人兴奋的发现是它们具有光活性——也就是说，它们的光学特性可以通过特定波长的光照射来控制。例如，绿色发射蛋白可以通过紫外线照射转化为红色发射体。这导致了“光学高光”，在空间的特定点产生不同颜色的蛋白质，并且可以将该群体的后续进化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蛋白，这些蛋白在照射之前是不荧光的（不同波长的光则相反）。这使得在任何时候只使少数蛋白质产生荧光成为可能。因此，极高对比度的单分子成像成为可能，允许超（纳米尺度）分辨率的蛋白质运动研究。这被称为第二次FP革命。我们的目标是了解我们称之为第二代FPs的光活性行为背后的光物理学。这个多学科项目得到了本地和国际合作的支持。揭示第二代FPs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，我们以低于50飞秒的分辨率记录发射强度和光谱的时间行为。这为激发态势能表面的分子动力学提供了独特的见解。这些动力学将作为氘化的函数来研究质子转移在光激活中的作用，研究蛋白质基质的诱变作用，研究pH值对不同残基滴定的影响，以及寻找光激活激发态能垒的存在和高度。这些数据将根据合作实验室正在进行的结构研究进一步解释。除了这些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FPs，这将允许更好地控制蛋白质的光物理特性。这些完整蛋白质的研究将由实验室合成的发色团单位的研究补充。对控制裸发色团光物理的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，也为测试发色团激发态势能表面的理论计算提供重要的基础数据。这项研究是必不可少的，因为需要更好的设计和更具体的FPs，不仅可以作为活细胞成像的探针，还可以作为光活性传感器分子，这将允许FPs用于绘制环境的位置和化学性质。这一目标的成功将大大扩大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.