Optically Resonant Nanotweezers

描述(由申请人提供)：在这项工作中，我们提出了一个由康奈尔大学Erickson和Chen实验室联合开展的项目，旨在展示一种全新的方法，通过开发单分子纳米光子光学捕获和荧光共振能量转移(FRET)技术来研究弱蛋白质-蛋白质相互作用。我们应用这项技术的分子系统是人类铜从细胞内铜伴侣HAH1到铜转运ATPase Wilson病蛋白(WDP)的转运途径。这一转运途径的功能异常可导致肝豆状核变性和家族性肌萎缩侧索硬化症等疾病。尽管它很重要，但关于HAH1和WDP如何相互作用的定量信息非常有限。获取这一信息的一个主要困难是缺乏一种单分子分析工具，它可以同时：(1)捕获并无限期地悬浮在自由溶液中的小分子；(2)有效地“集中”一组感兴趣的分子，使之能够研究弱的蛋白质-蛋白质相互作用；(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，我们计划在这里开发的核心技术进步是我们最近展示的光学共振纳米除草器。光学限制技术在单分子分析中的优势在于，它们可以在动态变化的背景溶液中悬浮和集中目标。然而，从根本上说，现有的光学限制技术受到衍射的限制，衍射将介质靶的尺寸限制在100 nm左右。我们在这里展示了我们的平面光学共振纳米挖掘器使我们能够以这样一种方式集中光能，从而增强这种力，从而捕获小到几纳米的分子，将我们带入使单一蛋白质测量成为可能的范围。在这项工作中，我们建议通过捕获一系列更大的测试蛋白(6-8 nm)来初步开发该系统，该系统建立在我们之前捕获核酸的工作基础上。在初步开发之后，我们将对Hah1-WDP络合物进行一系列单分子捕获-FRET研究，考察结合相互作用如何响应Cu1+离子本底浓度的变化。 与公众健康相关：金属离子，例如铁和铜，是必要的营养物质，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肝豆状核变性、门克斯病和家族性肌萎缩侧索硬化症等疾病。在这项工作中，我们建议开发一种全新的基于光学的单分子分析方法，并将其应用于了解一系列控制细胞内铜运输的蛋白质的功能。

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.