Optically Resonant Nanotweezers

光学共振纳米镊子

基本信息

批准号：
8069193
负责人：
David Carl Erickson
金额：
$ 17.82万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2013-04-30
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：在这项工作中，我们提出了康奈尔大学埃里克森和陈实验室之间的联合项目，旨在通过开发单分子纳米光子光捕获来展示一种研究弱蛋白质-蛋白质相互作用的全新方法和荧光共振能量转移（FRET）技术。我们在此应用该技术的分子系统是从细胞内铜伴侣 Hah1 到铜转运 ATP 威尔逊病蛋白 (WDP) 的人类铜转运途径。该转运途径的功能异常可导致威尔逊病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。获得此信息的一个主要困难是缺乏能够同时执行以下操作的单分子分析工具：(1) 在自由溶液中无限期地捕获和悬浮小分子 (2) 有效地将感兴趣的分子集“浓缩”到可以研究弱蛋白质-蛋白质相互作用的点，并且（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。光限制技术（如光镊）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浓缩目标。然而，从根本上讲，现有的光学限制技术受到衍射的限制，衍射对可捕获的电介质目标的尺寸设定了下限，约为 100nm。我们在这里证明，我们的平面光学谐振纳米镊子使我们能够集中光能，从而增强这种力，从而捕获小至几纳米的分子，使我们能够进行单一蛋白质测量。在这项工作中，我们建议在我们之前捕获核酸的工作基础上，通过捕获一系列较大的测试蛋白质（6-8nm）来初步开发该系统。初步开发后，我们将对 Hah1-WDP 复合物进行一系列单分子捕获 FRET 研究，检查结合相互作用如何响应 Cu1+ 离子背景浓度的变化。公共健康相关性：金属离子，例如铁和铜，是必需的营养素，如果其浓度超过生理极限，也可能有毒。金属转运分子功能异常可导致威尔逊病、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光学单分子分析方法，并将其应用于理解控制细胞内铜转运的一系列蛋白质的功能。