Single Molecule Enzymology with Carbon Nanocircuits

碳纳米电路的单分子酶学

• 批准号: 8115098
• 负责人: Gregory A. Weiss
• 金额: $ 22.44万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8115098
• 关键词: Affect; Architecture; Binding; Biochemical; Biological; Cancer Etiology; Carbon; Carbon Nanotubes; Catalysis; Caveolins; Cell physiology; Cells; Chemistry; Clinic; Coupling; Cysteine; Deoxyribonucleases; Development; Devices; Disease; Electrodes; Electron Transport; Electronics; Enzymatic Biochemistry; Enzymes; Event; Exhibits; Goals; Hereditary Disease; Individual; Kinetics; Laboratories; Learning; Link; Lysine; Malignant Neoplasms; Measurement; Mediating; Mediation; Methodology; Methods; Microfabrication; Microscope; Molecular; Monitor; Muramidase; Nanotubes; Proteins; Publishing; RNA Splicing; Reporting; Research Personnel; Science; Signal Transduction; Signaling Protein; Site; Sulfhydryl Compounds; Techniques; Thermodynamics; Time; Variant; Work; attenuation; base; carboxylate; caveolin 1; design; human disease; molecular dynamics; mutant; nanoscale; new technology; novel strategies; protein function; public health relevance; research study; single molecule; theories; tumor; tumorigenic

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.

描述（由申请人提供）：单个错误细胞可以引发癌症。为了引发这种疾病，突变蛋白会单独或成组地破坏正常的细胞功能。一种异常蛋白质是什么样的？突变体的动力学与野生型的动力学相比如何？在此提出的研究中，将单独检查单个分子，以表征它们对分子疾病的贡献的基础。用于一次检查蛋白质的显微镜是研究人员最近在《科学》杂志上报道的一种新型纳米电路。该项目利用了微加工和接触多个电极的单个碳纳米管的受控合成方面的进步。在发表的初步结果中，研究人员证明了在连接到纳米电路的纳米管的侧壁上引入了单个羧酸盐手柄的电导控制。通过与羧酸酯柄的生物共轭，单个蛋白质可以连接到纳米电路中。虽然标准 EDC/NHS 偶联化学提供了与随机赖氨酸的随机缀合，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。利用所得纳米电路的电子签名，将在蛋白质解折叠、折叠、结合以及（如果适用）催化过程中实时检查单个蛋白质。在具体目标 1 中，碳纳米电路的当前设计将扩展到并行多个蛋白质的灵敏测量。在下一个具体目标中探索的两个场景中，单分子实验将受益于这种并行设备架构。同时检测不同的蛋白质或蛋白质变体可以阐明相同条件下的功能差异，例如突变蛋白质与野生型蛋白质的异常。在下一个具体目标中，具体目标 1 中的碳纳米电路首先用于研究经过充分研究的蛋白质，从而为该方法建立基线。单分子酶学将探索电子转移、构象变化、变构和其他问题如何影响纳米电路电导。具体目标 3 扩展了第一个器件架构以及从第二个器件架构中学到的知识，以研究与癌症和其他疾病有关的小窝蛋白控制细胞信号传导的分子基础。拟议的研究探讨了小窝蛋白如何在一系列不同条件和突变变体下抑制不同的酶。总之，鉴于单分子事件对疾病诱发和传播的重要性，需要扩展单分子研究的方法。该应用程序利用研究人员实验室的最新进展来开发单分子酶学的通用方法。然后，将在单分子水平上探索小窝蛋白介导癌症的机制基础。 公共卫生相关性 单个蛋白质可以劫持细胞导致癌症和其他人类疾病。该项目开发了观察单个蛋白质的新技术。具体来说，将使用新型纳米级电子电路来研究小窝蛋白如何引导肿瘤形成。