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: 8016421
• 负责人: Adam Ezra Cohen
• 金额: $ 37.14万
• 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/8016421
• 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; 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）：将微生物视紫红质工程化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，能够响应膜电位的变化而发出光学信号，其目标是对体内神经元的电活动进行成像。这种分子还可用于探测线粒体、心肌细胞、细菌或其他非神经元细胞中的膜电位，从而为了解与人类健康和疾病有关的多种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称为绿色蛋白视紫红质（GPR）的微生物视紫红质蛋白。在野外，这种蛋白质吸收阳光并泵送质子以产生质子动力。我们将设计蛋白质使其反向运行，以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于单细胞成像来说具有足够的荧光。 GPR 可以在体外斑马鱼神经元和活体斑马鱼中表达和成像。 GPR 的单点突变导致蛋白质的荧光对膜电位极其敏感。 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的颜色决定官能团。当细胞处于静止状态时，该官能团被去质子化，并且蛋白质呈黑色。当细胞激发动作电位时，质子被迫进入该官能团，蛋白质变得明亮。 正如 GFP 通过追踪细胞中蛋白质位置的能力彻底改变了生物学一样，我们相信微生物视紫红质将通过其标记生物膜以及将膜电位转换为荧光变化的能力产生广泛的影响。 公共健康相关性：许多细胞膜维持跨膜电压差，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，可以提供膜电位的可见读数。这种蛋白质将有助于对与人类健康和疾病有关的多种细胞的电生理学研究。