Single Molecule Enzymology with Carbon Nanocircuits

描述（由申请人提供）：单个出错细胞可以诱发癌症。为了引发这种疾病，突变蛋白单独或成群地破坏正常的细胞功能。一个异常蛋白是什么样子的？突变体的动力学与野生型的动力学相比如何？在这里提出的研究中，单个分子将被单独检查，以表征它们对分子疾病的贡献的基础。研究人员最近在《科学》杂志上报道了一种新型的纳米电路，用于一次检查蛋白质。该项目利用了微加工技术的进步和单个碳纳米管接触多个电极的受控合成。在发表的初步结果中，研究人员展示了在连接到纳米电路的纳米管的侧壁上引入单个羧酸手柄的电导率控制。通过与羧酸柄的生物偶联，单个蛋白质可以连接到纳米电路中。虽然标准的EDC/NHS偶联化学提供随机偶联到随机赖氨酸，但特定的半胱氨酸游离硫醇可用于直接连接到蛋白质内的特定位点。利用合成纳米电路的电子签名，单个蛋白质将在蛋白质展开、折叠、结合和催化过程中进行实时检测。在具体目标1中，目前的碳纳米电路设计将扩展到多个蛋白质并行的敏感测量。单分子实验将受益于这种并行器件架构在两种情况下探讨的下一个具体目标。同时检测不同的蛋白质或蛋白质变体可以阐明在相同条件下的功能差异，例如突变蛋白与野生型的异常。在下一个特定目标中，来自specific aim 1的碳纳米电路首先被应用于研究已被充分研究的蛋白质，从而为该方法建立基线。单分子酶学将探讨电子转移、构象变化、变构和其他问题如何影响纳米电路的电导率。特异性Aim 3扩展了第一个和从第二个学到的设备架构，以研究与癌症和其他疾病有关的小窝蛋白控制细胞信号的分子基础。提出的研究考察了在一系列不同条件和突变变异下，小窝蛋白如何抑制不同的酶。综上所述，考虑到单分子事件对疾病引发和传播的重要性，需要扩展单分子研究方法。该应用程序利用调查员实验室的最新进展，开发了一种可推广的单分子酶学方法。然后，在单分子水平上探讨小窝蛋白介导癌症的机制基础。

DESCRIPTION (provided by applicant): A single errant cell can instigate cancer. To trigger this disease, mutant proteins either singly or in groups disrupt normal cellular function. What does one abnormal protein look like? How do the dynamics of the mutant compare to the kinetics of wild- type? In the studies proposed here, single molecules will be individually examined to characterize the basis for their contributions to molecular disease. The microscope used to examine the proteins one-at-a-time is a new type of nanocircuit reported by the Investigators recently in Science. The project leverages advances in microfabrication and the controlled synthesis of a single carbon nanotube contacting multiple electrodes. In published preliminary results, the Investigators have demonstrated conductance-controlled introduction of a single, carboxylate handle onto the sidewall of a nanotube connected into a nanocircuit. Through bioconjugation to the carboxylate handle, a single protein can be connected into the nanocircuit. Though standard EDC/NHS coupling chemistry provides stochastic conjugation to a random lysine, specific cysteine free thiols can be used to direct connections to particular sites within the protein. Using the electronic signature of the resultant nanocircuit, the single protein will be examined in real-time during protein unfolding, folding, binding, and, where applicable, catalysis. In Specific Aim 1, the current design for carbon nanocircuits will be extended for sensitive measurements with multiple proteins in parallel. Single molecule experiments will benefit from this parallel device architecture in two scenarios explored in the next specific aims. Simultaneous interrogation of different proteins or protein variants can elucidate functional differences under identical conditions, such as the abnormality of a mutant protein versus wild-type. In the next specific aim, the carbon nanocircuits from Specific Aim 1 are first applied to investigate well studied proteins, thus establishing a baseline for the approach. Single molecule enzymology will explore how electron transfer, conformational change, allostery, and other issues affect nanocircuit conductance. Specific Aim 3 extends device architectures from the first and what is learned from the second to investigate the molecular basis for caveolin control over cell signaling, implicated in cancer and other diseases. The proposed studies examine how caveolin inhibits different enzymes under a range of different conditions and mutational variants. In summary, given the importance of single molecule events to disease instigation and propagation, expanded methods for single molecule studies are needed. This application leverages recent advances from the Investigators laboratories to develop a generalizable approach for single molecule enzymology. Then, the mechanistic basis for caveolin mediation of cancer will be explored at the single molecule level. PUBLIC HEALTH RELEVANCE Individual proteins can hijack cells to cause cancer and other human diseases. This project develops new technologies for watching individual proteins. Specifically, how caveolin directs tumor formation will be investigated using a new type of nanometer-scale electronic circuit.