Single Molecule Optically Resonant NanoTweezers for the study of Intracellular Me

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开发一种新的方法来研究弱蛋白质-蛋白质相互作用，管理细胞内的金属和金属辅因子运输在单分子水平。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资助期间证明了这种镊子能够捕获小到几纳米的蛋白质，突破了光学物理学中长期存在的障碍。除了开发铜离子和维生素B12贩运的蛋白质相互作用动力学的全面信息，通过该计划，我们将开发两个通用的基于Nanotweezer的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的协议。金属分子伴侣介导金属离子和含金属辅因子在细胞内的安全运输以避免毒性，金属分子伴侣将这些反应性物质递送到它们的靶目的地，同时保护它们免受外源性毒性。 反应.这种转运途径的功能异常可导致疾病，如威尔逊病、门克斯病和家族性肌萎缩侧索硬化症。尽管其重要性，但关于使这种安全转移得以实现或导致其分解的生物物理机制的定量信息非常有限。获得该信息的主要困难是缺乏单分子分析工具，其可以同时：（1）捕获小分子并使其在游离溶液中悬浮不确定的时间段;（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。可以实现上述目标的一种潜在方法是通过使用光学镊子。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。利用光学共振纳米镊子技术，我们已经证明这种力可以增强1000倍，以便捕获小到几纳米的蛋白质（包括这里使用的威尔逊病蛋白质）。在这个提议中，我们展示了我们如何适应这种技术（1）非侵入性地捕获和悬浮游离溶液中的单个大分子（2）将其他分子引导到捕获区域，以便可以观察到相互作用，以及（3）在悬浮溶液改变时保持捕获的颗粒就位。当应用于细胞内金属转运时，这些能力可以加快发现metalochaperones如何响应不同环境条件以及最终导致上述病理的过程。

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.