All-optical molecular voltmeter for measuring electric fields in proteins

描述（由申请人提供）：这个项目是通过偶然的会议，并最终合作，三组。Rebane物理实验室对荧光染料的双光子吸收（2 PA）特性有着长期的研究计划，Hughes细胞生物学实验室已经在荧光蛋白和通道方面工作了二十年，Callis化学实验室对2 PA和蛋白质的静电特性有着深刻的理论理解。最初，该小组聚集在一起，以了解在荧光蛋白中形成2 PA的机制。这导致发现非常强的静电力正在塑造发色团的吸收特性。这一令人着迷的发现是生物学家、化学家和物理学家在各自学科的交叉点上相互融合的直接结果，这一交叉点已经成熟，可以进行探索。在这里，我们正在采取下一个合乎逻辑的步骤：确定我们是否以及如何使用2 PA的灵敏度来测量复杂蛋白质中的静电。如果成功的话，这项提议可能会导致一种新型的光学电压表，它可以以前所未有的准确度和保真度以全新的方式测量力。我们的方法的独特之处在于：7蛋白质中的场是在环境条件下测量的，实际上是在生命系统中; 7不依赖于外部场或任何类型的电接触; 7使用与现有光学显微镜兼容的全光学检测; 7使用近红外光操作，以最大限度地减少损伤并提供更深的样品穿透; 7使用近红外光操作，以最大限度地减少损伤并提供更深的样品穿透。在某些条件下，2 PA横截面是基态和激发态电偶极矩之差的函数，而电偶极矩之差又是作用在精确位置处的电场强度和方向的函数。我们利用2 PA截面的值和电场作用在发色团的位置在原位之间的定量关系。这是第一次允许从基本物理原理确定蛋白质，膜等内部的电场。这种方法的另一个吸引人的方面是，它与已经在许多生物研究实验室中战略定位的双光子显微镜很好地结合在一起。第一个具体的目标是开发一套参考荧光染料分子，在那里我们确切地知道分子的偶极矩依赖于从实验测量和量子化学计算的字段。在第二个目标中，我们将在简单的模型系统中测试我们的技术，例如将染料掺入市售蛋白质和人工脂质双层中。最后，我们将我们的探针共价连接到特定的半胱氨酸内的摇床钾通道和测量在不同的构象通道内的字段。我们正在与一个由光学和分子物理学、分子生物学和计算化学领域的国际知名专家组成的跨学科团队一起应对这些挑战。如果成功，该项目将开辟新的前沿，不仅在分子生物学和生物大分子的研究。由于其固有的简单性，我们预计我们的全光学分子电压表将是相对简单的使用，并可能适用于广泛的问题。 公共卫生相关性：我们提出了一种使用激光研究蛋白质分子静电特性的新方法。目标是观察这些分子如何在纳米尺度上在活细胞中发挥关键作用。这项新技术可以诊断和治疗由某些分子机制故障引起的疾病。

DESCRIPTION (provided by applicant): This project arose through the serendipitous meeting, and eventual collaboration, of three groups. The Rebane laboratory in physics has a long standing research program on two-photon absorption (2PA) properties of fluorescent dyes, the Hughes laboratory in cell biology has been working on fluorescent proteins and channels for two decades, and the Callis laboratory in chemistry has a deep theoretical understanding of 2PA and electrostatic properties of proteins. Initially the group came together to understand the mechanisms that shape 2PA in the fluorescent proteins. This led to the discovery that very strong electrostatic forces are shaping the absorption properties of the chromophore. This fascinating discovery was the direct result of an intermingling of biologists, chemists, and physicists at an intersection between their disciplines that was ripe for exploration. Here we are taking next logical step: determine if and how can we use the sensitivity of 2PA to measure electrostatics in complex proteins. If successful, the proposal may lead to a new kind of optical voltmeter that can measure forces in entirely new ways with unprecedented accuracy and fidelity. The unique requisites of our approach are: 7 The fields in proteins are measured under ambient conditions, indeed in living systems; 7 Not rely on external fields or any kind of electrical contacts; 7 Use all-optical detection compatible with existing optical microscopes; 7 Operate with near-infrared light to minimize damage and provide deeper sample penetration; Under certain conditions the 2PA cross section is a function of the difference between electrical dipole moment in ground- and excited state, which in turn is a function of electric field strength and direction acting at the precise location. We take advantage of quantitative relation between the value of 2PA cross section and the electric field acting at the location of the chromophore in situ. This allows, for the first time, determining the electric filed inside proteins, membranes etc. from basic physical principles. A further appealing aspect of this method is that it meshes well with two-photon microscopes already strategically positioned in many biology research labs. The first specific aim is to develop a set of reference fluorescent dyes molecules, where we know exactly the molecular dipole moment dependence on the field from experimental measurements and quantum-chemical calculations. In the second aim, we will test our technique in simple model systems such as dyes incorporated into commercially-available proteins and artificial lipid bi-layers. Finally, we will covalently attach our probes to specific cysteins within the shaker potassium channel and measure the field within the channel in different conformations. We are attacking these challenges with an interdisciplinary team comprising internationally renowned experts in optical- and molecular physics, molecular biology and computational chemistry. If successful, this project will open up new frontiers in not only in molecular biology and study of biological macromolecules. Due to its inherent simplicity, we anticipate that our all- optical molecular voltmeter will be relatively straightforward to use, and may be applied to a broad range of problems. PUBLIC HEALTH RELEVANCE: We are proposing a new way of studying electrostatic properties of protein molecules using laser light. The goal is to observe how such molecules perform key functions in living cells on a nanometer scale. This new technique may enable diagnosis and treatment of diseases caused by malfunction of certain molecular mechanisms.