Femtosecond to Millisecond Photo-dynamics of Third Generation Fluorescent Proteins

第三代荧光蛋白的飞秒至毫秒光动力学

• 批准号: EP/X011410/1
• 负责人: Stephen Meech
• 金额: $ 57.85万
• 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: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FX011410%2F1
• 关键词: Femtosecond; Millisecond; Photo; dynamics; Third

Optical microscopy has been central to biology for centuries. In recent decades fluorescence microscopy has developed as an exceptionally sensitive and universally employed tool in the life sciences. Its power was dramatically enhanced in the 1990s through the discovery of the green fluorescent protein (GFP). This allowed a fluorescent label (GFP) to be irreversibly and selectively expressed attached to a specific target protein, permitting the location, dynamics and function of that protein to be probed in a living cell. More recently, photoswitchable fluorescent proteins were discovered. These fluorescent proteins can be reversibly switched between fluorescent (on) and nonfluorescent (off) states by light. This property allowed the development of super-resolution fluorescence bioimaging, which improved the spatial resolution of fluorescence microscopy by more than 10 times, allowing details much smaller than the wavelength of light to be observed in living systems. However, applications of photoswitchable proteins are limited because the light used to generate fluorescence from their 'on' states also switches the proteins 'off', causing the image to fade. A higher level of control is offered by the recently discovered third generation fluorescent proteins (3G FPs), which have three states, an off-state and a switching state in thermal equilibrium with an on-state. These three states can be independently excited, so switching off is decoupled from observation from the on-state. This development will lead to enhanced super-resolution imaging, and has the potential for the development of new multicolour imaging methods. However, the mechanism connecting these states is completely unknown.Our experiments will allow us to probe in detail the effects of light on all three states of 3G FPs. We will measure the rates of the interconversion between the three states as well as the structural changes that accompany them. Since the fastest of the interconversion reactions are extremely fast (thousand-billionths of a second) we will use the tools of ultrafast laser spectroscopy to make our observations. Our ultrafast experiments probe populations of reactant, intermediate and product states through their absorption spectroscopy, while their structures are probed through ultrafast vibrational spectroscopy. These measurements will lead to a detailed picture of the photoconversion mechanism in 3G FPs. Once the mechanism is established, we will apply the tools of chemical biology to make mutations in key residues that will (i) test our ideas of the mechanism and (ii) optimise the photoswitching rate, and thus yield superior 3G FPs for bioimaging.Photoactive proteins such as FPs offer a unique opportunity to observe protein structure evolution in real time. In particular time resolved vibrational spectroscopy yields structural data from femtoseconds out to milliseconds. This information has great importance, as it can be compared with the results of computational calculations and independent experiments on protein structural dynamics. Together this will yield the most detailed insights yet into the dynamics of proteins undergoing their function, which in turn enhances our understanding of the nature of drug-protein interactions.

几个世纪以来，光学显微镜一直是生物学的核心。近几十年来，荧光显微镜已经发展成为生命科学中非常敏感和普遍使用的工具。在20世纪90年代，随着绿色荧光蛋白（GFP）的发现，它的能力得到了极大的增强。这使得荧光标记（GFP）能够不可逆地和选择性地表达，附着在特定的靶蛋白上，从而允许在活细胞中探测该蛋白的位置、动力学和功能。最近，发现了可光转换的荧光蛋白。这些荧光蛋白可以通过光在荧光（开）和非荧光（关）状态之间可逆地切换。这一特性使得超分辨率荧光生物成像技术得以发展，将荧光显微镜的空间分辨率提高了10倍以上，从而可以在生命系统中观察到比光波长小得多的细节。然而，光开关蛋白质的应用是有限的，因为用于从它们的“开”状态产生荧光的光也会将蛋白质“关”，导致图像褪色。最近发现的第三代荧光蛋白（3G FP）提供了更高水平的控制，其具有三种状态，即关闭状态和与打开状态热平衡的切换状态。这三种状态可以独立地被激发，因此关断与开状态的观察解耦。这一发展将导致增强的超分辨率成像，并有潜力开发新的多色成像方法。然而，连接这些状态的机制是完全未知的。我们的实验将使我们能够详细探测光对3G FP所有三种状态的影响。我们将测量这三种状态之间的相互转化率以及伴随它们的结构变化。由于最快的相互转换反应非常快（千分之十亿分之一秒），我们将使用超快激光光谱学工具进行观察。我们的超快实验通过它们的吸收光谱探测反应物，中间体和产物状态的种群，而它们的结构通过超快振动光谱探测。这些测量将导致3G FP中光转换机制的详细描述。一旦建立了机制，我们将应用化学生物学的工具，使关键残基的突变，将（i）测试我们的想法的机制和（ii）优化的光开关速率，从而产生上级3G FPs的生物成像。光敏蛋白质，如FPs提供了一个独特的机会，观察蛋白质结构的演变在真实的时间。特别是时间分辨振动光谱产生的结构数据从飞秒到毫秒。这些信息非常重要，因为它可以与蛋白质结构动力学的计算结果和独立实验进行比较。总之，这将产生最详细的见解，但蛋白质的动态进行其功能，这反过来又增强了我们对药物-蛋白质相互作用的性质的理解。