Computationally Driven Design of De Novo Genetically Encoded Voltage Indicators

项目总结： 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，将能够记录电路级的活动， 引发情感、行为和认知，并将推动对网络失调的新见解 潜在的精神和神经障碍。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)，是一种细胞类型的特定蛋白报告，可以转导 膜电位作为荧光信号。大规模采用地理信息系统有赖于它们拥有 亮度、电压敏感度和时间分辨率，允许在体内记录高频 脉冲串、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GEV，如由古紫质构建的GEV，并不具备所有这些所需的 属性，并在时间分辨率上受到内在限制，因为它们依赖于 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的地理信息系统 利用计算从头蛋白质设计中的有效方法来产生跨膜螺旋 结合近红外荧光的哺乳动物内源性胆绿素发色团的束蛋白。恰如其分 胆绿素在蛋白质中的定位将允许光学 斯塔克效应的电压报告，以及 一种固有的电压敏感现象，即作用于发色团的电场使 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 在正常和患病的大脑中可以看到复杂的表型。

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.