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

• 批准号: 8601119
• 负责人: David Carl Erickson
• 金额: $ 28.92万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 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/8601119
• 关键词: Affect; Binding; Biological Assay; Biological Availability; Biophysical Process; 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

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Erickson和Chen Labs之间的一个联合项目，以开发一种新方法来研究弱蛋白质 - 蛋白质相互作用，该蛋白质 - 蛋白质相互作用控制单分子水平的细胞内金属和金属co因子运输。该方法涉及使用“光学谐振纳米丝丝”，我们在探索性R21 Grant之前证明的，能够将蛋白质捕获至少几纳米，从而突破了光学物理学长期建立的障碍。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，通过该程序，我们还将开发两个基于纳米weezer的总体方案，用于定量的单分子氟氟[SMFQ）和单个分子氟氟此类（SMFRETENT），并将其用于许多其他许多其他问题。金属离子在细胞内的安全贩运金属离子和含金属辅助因子以避免毒性是由金属伴侣介导的，这些金属伴侣将这些反应性物种传递到其目标目的地，同时保护它们免受不定 反应。该运输途径的异常功能会导致疾病，例如威尔逊病，梅克斯病和家族性肌萎缩性侧面硬化症。尽管它很重要，但对于可以使这种安全转移或导致其分解的生物物理机制提供了非常有限的定量信息。获取此信息的主要困难是单个分子分析工具的湖泊，该工具可以同时进行：（1）在不确定的时间内（2）有效地“浓缩”感兴趣的分子集合较小的蛋白质相互作用，并可以研究外部环境条件的快速调节。可以实现上述目标的一种潜在方法是使用光学镊子。但是，从根本上讲，现有的光学限制技术受到衍射的限制，该衍射将介电靶标的大小放在可将其捕获到约100nm的介电靶标上。借助光学谐振的纳米韦策技术，我们已经表明，可以增强这种力量的1000次，以使蛋白质（包括此处使用的Wilson疾病蛋白质）和几纳米小纳米。在此提案中，我们展示了如何使该技术适应（1）在自由溶液中无创捕获和悬挂单个大分子（2）将附加分子引导到捕获区域，以便可以观察到相互作用，并且（3）将捕获的颗粒保持在位，而悬浮溶液则更改。当应用于细胞内金属传输时，这些能力可以加快发现金属伴侣如何响应不同环境条件的过程，并最终导致上述病理。