Single Molecule Optically Resonant NanoTweezers for the study of Intracellular Me

用于细胞内 Me 研究的单分子光学共振纳米镊子

基本信息

批准号：
8419345
负责人：
David Carl Erickson
金额：
$ 27.65万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-01-01 至 2016-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8419345
关键词：
Affect Binding Biological Assay Biological Availability Cells Complex Copper Destinations Development Disease Energy Transfer Environment Enzymes Familial Amyotrophic Lateral Sclerosis Goals Grant Hepatolenticular Degeneration Immobilization Individual Ions Joints Knowledge Lead Mediating Menkes Kinky Hair Syndrome Metabolism Metals Methods Methylmalonyl-CoA Mutase Molecular Chaperones Nutrient Optics Pathology Pathway interactions Phase Physics Physiological Positioning Attribute Process Protein Dynamics Proteins Protocols documentation Radial Reaction Series Solutions Speed System Techniques Technology Time Toxic effect Universities Vitamin B 12 Wilson disease protein Work base cobamamide cofactor interest laser tweezer macromolecule meetings nanometer nanovesicle novel strategies particle photonics programs protein complex protein protein interaction public health relevance research study single molecule small molecule success tool trafficking

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 develop a new approach to study the weak protein-protein interactions that govern intracellular metal and metal co-factor transport at the single molecule level. The approach involves the use of "Optically Resonant NanoTweezers" which we demonstrated during preceding exploratory R21 grant are capable of trapping proteins as small as a few nanometers, breaking through a long established barrier in optical physics. In addition to developing comprehensive information on the protein interaction dynamics for copper ion and vitamin B12 trafficking, through this program we will develop two general NanoTweezer based protocols for a quantitative single molecule florescence quenching assay (smFQ) and a single molecule florescence resonant energy transfer assay (smFRET) that can be applied to numerous other biophysical problems. Safe trafficking of metal ions and metal-containing cofactors inside cells to avoid toxicity is mediated by metallochaperones which deliver these reactive species to their target destinations while protecting them from adventitious reactions. Abnormal function of this transport pathway can lead to diseases such as Wilson disease, Menkes disease, and familial amyotrophic lateral sclerosis. Despite its importance, very limited quantitative information is available on the biophysical mechanisms that enable this safe transfer or cause it to break down. A major difficulty in obtaining this information is the lak 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. One potential method by which the above goals could be achieved is through the use of optical tweezers. 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. With the optically resonant nanotweezer technology we have shown that this force can be enhanced 1000's of times so as to trap proteins (including the Wilson disease proteins used here) as small as a few nanometers. In this proposal, we show how we can adapt this technology to (1) non-invasively capture and suspend individual macromolecules in free solution (2) guide additional molecules to the capture region so that interactions can be observed and (3) maintain captured particles in position while the suspending solution is changed. When applied to intracellular metal transport these capabilities can speed up the process for discovering how metalochaperones respond to different environmental conditions and ultimately what leads to the pathologies listed above.

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一个联合项目，开发一种新方法来研究控制细胞内金属和金属辅因子转运的弱蛋白质-蛋白质相互作用。分子水平。该方法涉及使用“光学共振纳米镊子”，我们在之前的探索性 R21 资助中证明了该镊子能够捕获小至几纳米的蛋白质，突破了光学物理学中长期存在的障碍。除了开发有关铜离子和维生素 B12 运输的蛋白质相互作用动力学的全面信息外，通过该计划，我们还将开发两种基于 NanoTweezer 的通用方案，用于定量单分子荧光猝灭测定 (smFQ) 和单分子荧光共振能量转移测定（smFRET）可应用于许多其他生物物理问题。金属离子和含金属辅助因子在细胞内的安全运输以避免毒性是由金属伴侣介导的，金属伴侣将这些活性物质传递到目标目的地，同时保护它们免受外来物质的影响反应。该转运途径的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。尽管它很重要，但关于实现这种安全转移或导致其分解的生物物理机制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。实现上述目标的一种潜在方法是使用光镊。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。通过光学共振纳米镊子技术，我们已经证明这种力可以增强数千倍，从而捕获小至几纳米的蛋白质（包括此处使用的威尔逊病蛋白质）。在本提案中，我们展示了如何采用该技术来（1）非侵入性地捕获并悬浮游离溶液中的单个大分子（2）将其他分子引导至捕获区域，以便可以观察到相互作用以及（3）维持捕获的颗粒更换悬浮液时就位。当应用于细胞内金属运输时，这些能力可以加快发现金属伴侣如何响应不同环境条件以及最终导致上述病理的过程。