Optically Resonant Nanotweezers

描述（由申请人提供）：在这项工作中，我们提出了一个联合项目之间的埃里克森和陈实验室在康奈尔大学，以证明一个全新的方法来研究弱蛋白质-蛋白质相互作用，通过发展单分子纳米光子光捕获和荧光共振能量转移（FRET）技术。我们应用该技术的分子系统是从细胞内铜伴侣Hah 1到铜转运ATP酶Wilson病蛋白（WDP）的人类铜转运途径。这种转运途径的功能异常可导致疾病，如威尔逊病和家族性肌萎缩侧索硬化症。尽管它的重要性，非常有限的定量信息是如何Hah 1和WDP相互作用。获得这一信息的主要困难是缺乏一种单分子分析工具，它可以同时：（1）捕获小分子并将其悬浮在自由溶液中不确定的时间段（2）有效地将感兴趣的分子组“浓缩”到可以研究弱蛋白质-蛋白质相互作用的点，以及（3）允许快速调节外部环境条件（例如背景离子浓度）。为了满足这些要求，我们建议利用的核心技术进步是我们最近展示的光学谐振纳米镊子。光学限制技术的优势，如光镊，在单分子分析是，他们可以暂停和集中目标在动态变化的背景解决方案。然而，从根本上说，现有的光学限制技术受到衍射的限制，衍射对可以被捕获到约100 nm的电介质目标的尺寸设置了下限。我们在这里证明，我们的平面光学共振纳米镊子使我们能够集中光能，以这种方式，这种力可以增强，以捕获分子小到几纳米，使我们进入范围，使单一蛋白质测量成为可能。在这项工作中，我们建议通过捕获一系列更大的测试蛋白质（6- 8 nm）来初步开发该系统，该系统建立在我们以前捕获核酸的工作基础上。初步开发后，我们将进行一系列的单分子捕获FRET研究的Hah 1-WDP复杂的检查如何结合相互作用响应Cu 1+离子背景浓度的变化。 公共卫生相关性：金属离子，例如铁和铜，是必需的营养素，如果它们的浓度超过生理极限，也可能是有毒的。金属转运分子功能异常可导致Wilson病、Menkes病和家族性肌萎缩侧索硬化等疾病。在这项工作中，我们提出了一个全新的方法，以光学为基础的单分子分析，并将其应用于了解一系列蛋白质的功能，控制细胞内铜转运。

DESCRIPTION (provided by applicant): In this work, we propose a joint project between the Erickson and Chen labs at Cornell University to demonstrate an entirely new approach to the study of weak protein-protein interactions through the development of a single molecule nanophotonic optical trapping and florescence resonant energy transfer (FRET) technique. The molecular system we apply the technique to here is the human copper transport pathway from the intracellular copper chaperone Hah1 to the copper transporting ATPase Wilson disease protein (WDP). Abnormal function of this transport pathway can lead to diseases such as Wilson disease and familial amyotrophic lateral sclerosis. Despite its importance, very limited quantitative information is available on how Hah1 and WDP interact. A major difficulty in obtaining this information is the lack of a single molecule analysis tool which can simultaneously: (1) capture and suspend small molecules in free solution for an indefinite period time (2) effectively "concentrate" the set of molecules of interest to a point where weak protein-protein interactions can be studied and (3) allow rapid modulation of the external environmental conditions (e.g. background ion concentration). The core technological advancement we propose to exploit here in order to meet these requirements is our recently demonstrated optically resonant nanotweezers. The advantage of optical confinement techniques, like optical tweezers, in single molecule analysis is that they can suspend and concentrate targets in dynamically changing background solutions. Fundamentally however, existing optical confinement techniques are limited by diffraction which places a lower bound on the size of dielectric target which can be trapped to about 100nm. We demonstrate here that our planar optically resonant nanotweezers allow us to concentrate the optical energy in such a way that this force can be enhanced so as to trap molecules as small as a few nanometers, bringing us down into the range to make single protein measurements possible. In this work we propose to initially develop the system by trapping a series of larger test proteins (6-8nm) building on our previous work in trapping nucleic acids. After initial development we will conduct a series of single molecule trapping-FRET studies on the Hah1-WDP complex examining how binding interactions respond to changes in Cu1+ ion background concentration. PUBLIC HEALTH RELEVANCE: Metal ions, for example iron and copper, are essential nutrients that can also be toxic if their concentration exceeds the physiological limit. Abnormal function of metal transport molecules can lead to diseases such as Wilson disease, Menkes disease and familial amyotrophic lateral sclerosis. In this work we propose to develop a fundamentally new approach to optically based single molecule analysis and apply it to understanding the function of a series of proteins which control intracellular copper transport.