Engineering Microbial Rhodopsins as Optical Voltage Sensors

将微生物视紫红质工程化为光学电压传感器

• 批准号: 8588923
• 负责人: Adam Ezra Cohen
• 金额: $ 35.96万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-15 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8588923
• 关键词: Action Potentials; Axon; Bacteria; Bacteriorhodopsins; Biological; Biology; Blinking; Cardiac; Cell membrane; Cells; Codon Nucleotides; Color; Communication; Custom; Disease; Dreams; Dyes; Electrophysiology (science); Engineering; Erythrocytes; Escherichia coli; Fire - disasters; Fluorescence; Generations; Genetic; Goals; Halorhodopsins; Health; Human; Image; In Vitro; Integral Membrane Protein; Label; Lead; Libraries; Life; Light; Measures; Membrane; Membrane Potentials; Microbial Rhodopsins; Mitochondria; Molecular Probes; Mutagenesis; Neuroglia; Neurons; Optics; Physiological; Point Mutation; Positioning Attribute; Property; Protein Engineering; Proteins; Proton Pump; Proton-Motive Force; Protons; Relative (related person); Rest; Retinal; Rhodopsin; Running; Signal Transduction; Speed; Sunlight; System; Time; Work; Zebrafish; absorption; analog; base; cellular imaging; chromophore; design; functional group; improved; in vivo; insight; mutant; novel; public health relevance; quantum; response; sensor; sensory rhodopsin I; tool; voltage

DESCRIPTION (provided by applicant): Engineering Microbial Rhodopsins as Optical Voltage Sensors Neuroscientists have long dreamed of a genetically encoded sensor that gives an optical signal in response to a change in membrane potential, with the goal of imaging electrical activity of neurons in vivo. Such a molecule could also be used to probe membrane potentials in mitochondria, cardiac cells, bacteria, or in other non-neuronal cells, and thus would provide a new window into the physiological states of a wide range of cells implicated in human health and disease. We propose to engineer a fluorescent transmembrane protein whose fluorescence is sensitive to membrane potential. The goal is to visualize a single action potential in vivo. Many groups have sought to attain this goal; our approach is entirely different from previous efforts. Our starting material is a microbial rhodopsin protein called green proteorhodopsin (GPR). In the wild, this protein absorbs sunlight and pumps protons to generate a proton motive force. We will engineer the protein to run backward-to use membrane voltage to modulate light. The retinal chromophore in wild-type microbial rhodopsins is sufficiently fluorescent for single-cell imaging. GPR can be expressed and imaged in zebra fish neurons in vitro and in living zebra fish. A single-point mutation to GPR leads to a protein whose fluorescence is exquisitely sensitive to membrane potential. The essence of the idea is to use membrane potential to pull a proton toward or away from a color- determining functional group in the protein. When the cell is at rest, this functional group is deprotonated and the protein is dark. When the cell fires an action potential, a proton is forced onto this functional group and the protein becomes bright. Just as GFP revolutionized biology through its ability to track the positions of proteins in cells, we believe that microbial rhodopsins will have a broad impact through their ability to label biological membranes, and to transduce membrane potential into changes in fluorescence. PUBLIC HEALTH RELEVANCE: Many cell membranes maintain a voltage difference across the membrane, which is used for communication (in neurons), and for generation of energy (in bacteria and mitochondria). Our goal is to develop a protein that when expressed in a cell gives a visible readout of the membrane potential. This protein will facilitate studies on the electrophysiology of a wide range of cells implicated in human health and disease.

描述（由申请人提供）：工程微生物视紫红蛋白作为光电压传感器神经科学家长期以来梦想着一种遗传编码的传感器，该传感器响应于膜电位变化，其目的是将神经元的电活动成像。这种分子也可以用于探测线粒体，心脏细胞，细菌或其他非神经元细胞中的膜电位，因此将为与人类健康和疾病有关的广泛细胞的生理状态提供新的窗口。 我们建议设计一种荧光跨膜蛋白，其荧光对膜电位敏感。目的是在体内可视化单个动作电位。许多团体试图实现这一目标。我们的方法与以前的努力完全不同。 我们的起始材料是一种称为绿色蛋白质蛋白质（GPR）的微生物视紫红质蛋白。在野外，该蛋白质吸收阳光并泵送质子以产生质子动力。我们将设计蛋白质以向后运行，以使用膜电压调节光。野生型微生物视紫红质中的视网膜发色团足够荧光，用于单细胞成像。 GPR可以在体外和活着的斑马鱼中表达和成像。 GPR的单点突变导致蛋白质，其荧光对膜电位非常敏感。 该想法的本质是利用膜电位将质子拉向或远离蛋白质中的颜色确定官能团。当细胞处于静止状态时，该功能组将被质子化，并且蛋白质是黑暗的。当细胞发射动作电位时，将质子强加于该功能基团，并且蛋白质变得明亮。 正如GFP通过跟踪细胞中蛋白质位置的能力彻底改变了生物学一样，我们认为微生物视紫红蛋白将通过标记生物膜并将膜电位转换为荧光变化的能力，从而产生广泛的影响。 公共卫生相关性：许多细胞膜在整个膜上保持电压差，用于通信（在神经元中）和产生能量（在细菌和线粒体中）。我们的目标是开发一种蛋白质，当在细胞中表达时，可以对膜电位进行可见的读数。该蛋白质将促进有关与人类健康和疾病有关的广泛细胞的电生理学研究。