Computationally Driven Design of De Novo Genetically Encoded Voltage Indicators

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比， 引起了情感、行为和认知，并将推动对网络失调的新见解 潜在的精神和神经疾病。因此，在设计上花费了巨大的努力， 基因编码的电压指示器（GEVI），细胞类型特异性蛋白质报告， 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，以允许在体内记录高频 爆发，监测阈下电压动态，并准确重建动作电位 波形目前的GEVI，例如由古视紫红质构建的GEVI，不具有所有这些期望的功能。 由于它们依赖于结构， 重排的信号转导。我们建议建设高时间分辨率GEVI的 利用经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、胆甾体内源性胆绿素发色团的束蛋白。适当 胆绿素在蛋白质内的定位将允许光学 电压报告的斯塔克效应， 本征电压敏感现象，其中作用于发色团的电场引起亚- 通过改变吸光度使荧光发生皮秒变化。这项工作将产生近红外荧光， 高时间分辨率电压探头允许新的见解电路水平的生理基础 在正常和患病大脑中观察到的复杂表型。

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.