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: 8401906
• 负责人: Adam Ezra Cohen
• 金额: $ 34.98万
• 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/8401906
• 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.

描述（由申请人提供）：工程微生物视紫红质作为光学电压传感器神经科学家长期以来一直梦想着一种基因编码传感器，它可以响应膜电位的变化而发出光学信号，目的是成像体内神经元的电活动。这种分子也可以用来探测线粒体、心脏细胞、细菌或其他非神经元细胞的膜电位，从而为了解与人类健康和疾病有关的各种细胞的生理状态提供了一个新的窗口。我们拟设计一种荧光跨膜蛋白，其荧光对膜电位敏感。目标是可视化体内单个动作电位。许多团体都在努力实现这一目标；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叫做绿色变形紫红质（GPR）的微生物紫红质蛋白。在野外，这种蛋白质吸收阳光并泵送质子以产生质子动力。我们将设计这种蛋白质反向运行——利用膜电压来调制光。野生型微生物视紫红质中的视网膜发色团对单细胞成像具有足够的荧光。GPR在体外和活体斑马鱼神经元中均有表达和成像。GPR的单点突变导致一种蛋白质的荧光对膜电位非常敏感。这个想法的本质是利用膜电位将质子拉向或远离蛋白质中决定颜色的官能团。当细胞处于静止状态时，这个官能团去质子化，蛋白质变暗。当细胞发出动作电位时，一个质子被强制进入这个官能团，蛋白质就会变亮。正如绿色荧光蛋白通过其追踪细胞中蛋白质位置的能力彻底改变了生物学一样，我们相信微生物紫红质将通过其标记生物膜的能力产生广泛的影响，并将膜电位转化为荧光的变化。