All-optical molecular voltmeter for measuring electric fields in proteins

用于测量蛋白质电场的全光学分子伏特计

• 批准号: 8075226
• 负责人: Aleksander Rebane
• 金额: $ 24.24万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8075226
• 关键词: Amino Acids; Biological; Biological Models; Biology; Burn injury; Cell surface; Cells; Cellular biology; Charge; Chemicals; Chemistry; Collaborations; Cysteine; Data; Dependence; Detection; Diagnosis; Discipline; Disease; Drops; Dyes; Electrostatics; Environment; Fluorescein; Fluorescence; Fluorescent Dyes; Fluorescent Probes; Fluorometry; Goals; In Situ; Kinetics; Laboratories; Lasers; Lead; Letters; Life; Light; Link; Lipid Bilayers; Lipids; Location; Maleimides; Measurement; Measures; Membrane Proteins; Methods; Microscope; Modeling; Molecular; Molecular Biology; Molecular Conformation; Movement; Optics; Organic Synthesis; Organism; Penetration; Peptides; Physics; Positioning Attribute; Potassium Channel; Property; Proteins; Relative (related person); Research; Rhodamine; Sampling; Shaker potassium channel; Shapes; Site; Solvents; Spectrum Analysis; Sulfhydryl Compounds; System; Techniques; Testing; Tetrapyrroles; Time; Vacuum; Vesicle; Work; Xenopus oocyte; absorption; chromophore; computational chemistry; cyanine dye 5; dipole moment; electric field; fascinate; frontier; macromolecule; meetings; nanoscale; programs; protein complex; quantum; research study; sensor; simulation; two-photon; vector; voltage clamp

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.

描述(由申请人提供)：这个项目是由三个小组偶然的会面和最终的合作产生的。Rebane物理实验室对荧光染料的双光子吸收(2PA)特性有长期的研究计划，休斯细胞生物学实验室20年来一直致力于荧光蛋白质和通道的研究，而Callis化学实验室对2PA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塑造2PA的机制。这导致了发现，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，这一学科的探索时机已经成熟。在这里，我们采取下一个合乎逻辑的步骤：确定我们是否以及如何使用2PA的灵敏度来测量复杂蛋白质中的静电性。如果成功，这项提议可能会导致一种新型的光学伏特计，它可以以前所未有的精度和保真度以全新的方式测量力。我们这种方法的独特要求是：7在环境条件下，甚至在生命系统中测量蛋白质中的场；7不依赖外场或任何形式的电接触；7使用与现有光学显微镜兼容的全光学探测；7使用近红外光操作，以将损害降至最低，并提供更深的样品穿透；在某些条件下，2pa的横截面是基态和激发态的电偶极矩之差的函数，这反过来又是精确位置的电场强度和方向的函数。我们利用了2pA截面值与作用于发色团位置的电场之间的定量关系。这使得第一次能够从基本的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处于战略地位的双光子显微镜很好地结合在一起。第一个具体目标是开发一组参考荧光染料分子，其中我们通过实验测量和量子化学计算准确地知道分子偶极矩与场的关系。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，例如加入到商业可获得的蛋白质和人造脂质双层中的染料。最后，我们将我们的探针共价连接到摇床钾通道内的特定半胱氨酸上，并测量不同构象的通道内的场。我们正在与一个由国际知名的光学和分子物理、分子生物学和计算化学专家组成的跨学科团队来应对这些挑战。如果成功，该项目不仅将在分子生物学和生物大分子研究方面开辟新的前沿。由于其固有的简单性，我们预计我们的全光分子电压计将相对简单地使用，并可应用于广泛的问题。 与公共健康相关：我们提出了一种利用激光研究蛋白质分子静电特性的新方法。其目标是在纳米尺度上观察这些分子如何在活细胞中发挥关键功能。这项新技术可能使诊断和治疗由某些分子机制故障引起的疾病成为可能。