Computationally Driven Design of De Novo Genetically Encoded Voltage Indicators

De Novo 基因编码电压指示器的计算驱动设计

基本信息

批准号：
10556358
负责人：
Ivan Kuznetsov
金额：
$ 1.59万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2023-05-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10556358
关键词：
Action Potentials Address Adoption Affect Affinity Behavior Biliverdine Binding Binding Proteins Biochemistry Brain Diseases Calibration Cells Cognition Complex Directed Molecular Evolution Electric Capacitance Electrophysiology (science)Engineering Escherichia coli Exhibits Film Fluorescence Fluorescence-Activated Cell Sorting Frequencies Hippocampus Image In Vitro Kinetics Ligands Lipid Bilayers Lyase Mammalian Cell Measures Membrane Membrane Potentials Membrane Protein Traffic Mental disorders Methods Modeling Monitor Neurons Optics Phenotype Physiology Positioning Attribute Property Protein Engineering Proteins Protocols documentation Reporter Reporting Resistance Sampling Side Signal Transduction Spectrum Analysis Study models Thinness Tissue imaging Tissues Transfection Variant Viral Work Yeasts cell type chromophore design dichroism dipole moment electric field fluorescence imaging improved in vivo insight nervous system disorder neuroimaging novel patch clamp phycobilin reconstruction response success temporal measurement trafficking voltage

项目摘要

PROJECT SUMMARY: Simultaneous optical monitoring of the electrical activity of hundreds of neurons with single-cell fidelity comparable to whole-cell patch clamp electrophysiology would enable recording of the circuit-level activity that gives rise to affect, behavior, and cognition and would drive novel insights into the network dysregulation underlying psychiatric and neurological disorders. Therefore, enormous effort has been expended on designing genetically encoded voltage indicators (GEVIs), cell-type specific protein reporters that transduce changes in membrane potential as a fluorescent signal. Large-scale adoption of GEVIs is dependent on them possessing the brightness, voltage-sensitivity, and temporal resolution to allow for the in vivo recording of high frequency bursts, the monitoring of sub-threshold voltage dynamics, and the accurate reconstruction of action potential waveforms. Current GEVIs, such as those constructed from archaerhodopsin, do not possess all these desired properties and are intrinsically limited in their temporal resolution due to their reliance on structural rearrangements for signal transduction. We propose the construction of high temporal resolution GEVIs by leveraging validated methods in computational de novo protein design to produce transmembrane helical bundle proteins that bind a near-infrared fluorescent, mammalian-endogenous biliverdin chromophore. Proper positioning of biliverdin within the protein will allow for optical voltage-reporting by the Stark effect, an intrinsically voltage-sensitive phenomenon wherein an electric field acting upon a chromophore causes sub- picosecond changes in fluorescence by altering absorbance. This work will produce near-infrared fluorescent, high temporal resolution voltage probes permitting new insights into the circuit-level physiology underlying the complex phenotypes seen in the normal and diseased brain.

项目概要：以单细胞保真度同时光学监测数百个神经元的电活动与全细胞膜片钳电生理学相当，可以记录电路级活动产生情感、行为和认知，并将推动对网络失调的新见解潜在的精神和神经系统疾病。因此，在设计上花费了巨大的精力基因编码电压指示器（GEVI），细胞类型特异性蛋白质记者，可转导膜电位作为荧光信号。 GEVI 的大规模采用取决于它们拥有亮度、电压灵敏度和时间分辨率，可实现高频的体内记录突发、亚阈值电压动态监测以及动作电位的准确重建波形。目前的 GEVI，例如由古视紫红质构建的 GEVI，不具备所有这些所需的功能。由于其对结构的依赖，其时间分辨率本质上受到限制信号转导的重排。我们建议通过以下方式构建高时间分辨率 GEVI：利用经过验证的计算从头蛋白质设计方法来产生跨膜螺旋结合近红外荧光、哺乳动物内源性胆绿素发色团的束蛋白。恰当的胆绿素在蛋白质内的定位将允许光学斯塔克效应的电压报告，本质上的电压敏感现象，其中作用于发色团的电场导致子通过改变吸光度来改变荧光的皮秒变化。这项工作将产生近红外荧光，高时间分辨率电压探头允许对潜在的电路级生理学有新的见解在正常和患病大脑中看到的复杂表型。