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，我们提出了康奈尔大学Erickson和Chen实验室的一个联合项目，以开发一种在单分子水平上研究控制细胞内金属和金属辅助因子运输的弱蛋白质-蛋白质相互作用的新方法。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资助中演示过，能够捕获小至几纳米的蛋白质，突破了光学物理学中长期建立的障碍。除了开发铜离子和维生素B12运输的蛋白质相互作用动力学的综合信息外，通过该计划，我们将开发两种基于纳米weezer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。细胞内金属离子和含金属辅助因子的安全运输是由金属伴侣介导的，金属伴侣将这些活性物质运送到目标目的地，同时保护它们免受外来因素的影响

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.