Single Molecule Enzymology with Carbon Nanocircuits

碳纳米电路的单分子酶学

基本信息

批准号：
8305167
负责人：
Gregory A. Weiss
金额：
$ 22.16万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
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/8305167
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

项目摘要

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）：单个错误的细胞可以激发癌症。为了触发这种疾病，突变蛋白要么单独或组中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动力学相比如何？在此处提出的研究中，将对单分子进行单独检查，以表征其对分子疾病的贡献的基础。用于检查蛋白质单时检查蛋白质的显微镜是研究人员最近在科学领域报道的一种新型纳米电路。该项目利用了微加工的进步以及单个碳纳米管接触多个电极的受控合成。在已发表的初步结果中，研究人员证明了电导控制的羧酸盐手柄在连接到纳米电路的纳米管的侧壁上引入。通过与羧酸盐手柄的生物结合，可以将单个蛋白质连接到纳米电路中。尽管标准的EDC/NHS耦合化学为随机赖氨酸提供随机结合，但特定的无半胱氨酸硫醇可用于将连接与蛋白质内的特定位点进行连接。使用所得纳米电路的电子特征，将在蛋白质展开，折叠，结合以及适用的情况下实时检查单蛋白。在特定的目标1中，将扩展当前的碳纳米电路设计，以平行使用多种蛋白质进行敏感测量。单分子实验将受益于在下一个特定目标中探索的两种情况下的平行设备体系结构。同时询问不同蛋白质或蛋白质变体可以在相同条件下阐明功能差异，例如突变蛋白与野生型的异常。在下一个特定目的中，首先将来自特定目标1的碳纳米回路应用于研究良好的蛋白质，从而建立了该方法的基线。单分子酶学将探讨电子传递，构象变化，变构和其他问题如何影响纳米电路电导。特定的目标3扩展了设备架构从第一个延伸，以及从第二个学到的知识，以研究可爱素对细胞信号传导的分子基础，这与癌症和其他疾病有关。拟议的研究检查了小窝蛋白如何在各种不同条件和突变变异的范围内抑制不同的酶。总而言之，鉴于单分子事件对疾病爆发和传播的重要性，需要扩展单分子研究的方法。该应用利用研究人员实验室的最新进展来开发一种可推广的单分子酶学方法。然后，将在单分子水平上探索可爱蛋白介导的小窝蛋白介导的机械基础。公共卫生相关的单个蛋白质可以劫持细胞引起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。具体而言，将使用新型的纳米尺度电子电路研究小窝蛋白如何指导肿瘤形成。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Single-molecule dynamics of lysozyme processing distinguishes linear and cross-linked peptidoglycan substrates.

DOI：
10.1021/ja211540z
发表时间：
2012-02-01
期刊：
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
影响因子：
15
作者：
Choi, Yongki;Moody, Issa S.;Sims, Patrick C.;Hunt, Steven R.;Corso, Brad L.;Seitz, David E.;Blaszcazk, Larry C.;Collins, Philip G.;Weiss, Gregory A.
通讯作者：
Weiss, Gregory A.

Observing lysozyme's closing and opening motions by high-resolution single-molecule enzymology.

DOI：
10.1021/cb500750v
发表时间：
2015-06-19
期刊：
ACS chemical biology
影响因子：
4
作者：
Akhterov MV;Choi Y;Olsen TJ;Sims PC;Iftikhar M;Gul OT;Corso BL;Weiss GA;Collins PG
通讯作者：
Collins PG

Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase I (Klenow Fragment) Nanocircuits.