Photodynamics in Second Generation Fluorescent Proteins

第二代荧光蛋白的光动力学

• 批准号: EP/H025715/1
• 负责人: Stephen Meech
• 金额: $ 44.6万
• 依托单位: University of East Anglia
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FH025715%2F1
• 关键词: Photodynamics; Second; Generation; Fluorescent; Proteins

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.

从一种相对不起眼的水母中分离出的一种天然荧光蛋白（FP）可以在其他生物中克隆和表达，这一发现导致了生物成像的革命性进展。将绿色荧光蛋白的基因剪接到感兴趣的蛋白的基因确保了每当靶蛋白表达时，它与其荧光配偶体不可逆地结合。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，因为它是创建的，执行其功能并最终降解。在这个最初的发现之后不久，产生了几种FP的突变体，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-蛋白质相互作用。几年前，FP家族的另一个分支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敏的-也就是说它们的光学特性可以通过用特定波长的光照射来操纵。例如，发射绿色的蛋白质可以通过UV照射转化为红色发射体。这导致了“光学突出显示”，其中不同颜色的蛋白质在空间中的特定点产生，并且该群体的后续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活化的蛋白质，这些蛋白质在照射之前是不发荧光的（对于不同波长的光，发生相反的过程）。这使得在任何时候只有少数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，允许蛋白质运动的超（纳米级）分辨率研究。这被称为第二次FP革命。我们的目标是了解我们所称的第二代FP的光活性行为背后的电子物理学。这个跨学科的计划得到本地和国际合作的支持。解开第二代FP激发态化学的主要工具是荧光，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。这些动力学将被研究作为一个功能的氘化解开质子转移的作用，在光活化，诱变研究的蛋白质基质的作用，pH值探测滴定不同的残基和温度的影响，寻找激发态能量障碍的存在和高度的光活化。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。除了天然蛋白质及其突变体的这些研究，我们将扩大我们的调查含有非天然氨基酸的FP，这将允许更好地控制蛋白质的生物物理性质。这些完整蛋白质的研究将通过实验室合成的发色团单元的研究来补充。控制裸发色团的光物理学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，因为需要更好地设计和更具体的FP，不仅作为活细胞成像的探针，而且作为光敏传感器分子，这将允许FP用于绘制出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。

项目成果

Ultrafast proton transfer in the green fluorescent protein: Analysing the instantaneous emission at product state wavelengths

绿色荧光蛋白中的超快质子转移：分析产物状态波长下的瞬时发射

• DOI: 10.1016/j.jphotochem.2011.12.016
• 发表时间: 2012
• 期刊: Chemistry
• 作者: Kondo M

Excited state structural dynamics in higher lying electronic states: S2 state of malachite green

• DOI: 10.1016/j.cplett.2014.05.050
• 发表时间: 2014-06
• 期刊: Chemical Physics Letters
• 影响因子: 2.8
• 作者: S. Laptenok;K. Addison;I. Heisler;S. Meech

Complexation of Green and Red Kaede Fluorescent Protein Chromophores by a Zwitterion to Probe Electrostatic and Induction Field Effects.

绿色和红色Kaede荧光蛋白发色团的络合通过s扭转，以探测静电和诱导场效应。

• DOI: 10.1021/acs.jpca.1c10628
• 发表时间: 2022-02-24
• 期刊: JOURNAL OF PHYSICAL CHEMISTRY A
• 影响因子: 2.9
• 作者: Ashworth, Eleanor K.;Stockett, Mark H.;Kjaer, Christina;Page, Philip C. Bulman;Meech, Stephen R.;Nielsen, Steen Brondsted;Bull, James N.
• 通讯作者: Bull, James N.

Photophysics of the red-form Kaede chromophore.

• DOI: 10.1039/d3sc00368j
• 发表时间: 2023-04-05
• 期刊: Chemical science
• 影响因子: 8.4