Engineering high-performance voltage sensors for optogenetic imaging of neuronal circuit activity

1134416 Lin能够控制和监测神经元活动对于学习神经元回路如何处理信息和做出决策至关重要。然而，尽管最近已经开发出了使用基因编码的光激活通道来控制神经元放电的强大工具，但目前神经元活动的基因编码传感器的局限性严重限制了它们用于监测脑功能。特别是，钙传感器速度慢，不能报告阈下去极化。虽然电压传感器原则上可用于跟踪动作电位的快速序列并监测亚阈值去极化，但现有传感器的低动态范围使其不适合大多数体内实验。此外，目前没有钙或电压传感器被红色（550 nm）波长有效地激发，该提案的目标是通过开发新一代基因编码的电压生物传感器来满足这些需求，该生物传感器具有显着改善的动态范围和速度以及与光同时使用的能力-激活渠道这项工作将使神经系统的功能解剖更加精细和强大，同时也建立了新的蛋白质工程方法。实验室在荧光蛋白工程方面的专业知识将被用来教育未来的科学家和工程师关于蛋白工程的研究，并向教育工作者介绍基因编码生物传感器在实验室课程中教授科学概念的实用性。智力优势本提案的工程目标是应用荧光光谱和蛋白质结构的知识，以定量地改善现有一类生物传感器的性能。电压传感器（目标1）和开发一种全新的电压传感器（目标2）。在第一个目标中，我们将通过合理优化连接到电压传感结构域的荧光蛋白之间的Förster共振能量转移（FRET）来增加荧光范围。在第二个目标中，我们将使用构象敏感的荧光蛋白报告具有快速动力学的电压传感域的运动。为了达到这两个目的，我们将采用合理的设计，结合全面的饱和诱变和重要位点的筛选。这将是第一个应用Förster共振能量转移模型预测来确定限制荧光输出响应的因素，然后进行蛋白质工程以全面解决这些因素。第二，本项目将首次将联合收割机综合筛选与传感结构域和荧光蛋白的原子级结构知识相结合，以创建一种全新的变构电压传感器;因此，这项研究将使人们深入了解基因编码生物传感器工程的先验知识和筛选的相对有用性。更广泛的影响通过本项目开发的电压传感器将迅速传播到更大的科学界。这些传感器将对神经科学领域产生广泛的影响，使鲁棒的电压传感和并发控制和读出神经元活动。我们的工作也将影响生物工程领域，通过验证概念和建立策略来优化基于荧光蛋白的传感器的设计，利用能量转移或变构。拟议的项目将提供一个很好的机会，介绍本科生和高中生，包括来自弱势背景的学生，在分子生物工程的研究。它还将提供机会，向教育工作者介绍遗传编码生物传感的使用，以创建令人兴奋的实验室课程，教授重要的基本概念，同时展示蛋白质工程的最新研究成果。

1134416LinBeing able to both control and monitor neuronal activity is critical for learning how neuronalcircuits process information and make decisions. However, while powerful tools to control neuronal firingusing genetically encoded light-activated channels have recently been developed, limitations of currentgenetically encoded sensors of neuronal activity severely restrict their use for monitoring brain function.In particular, calcium sensors are slow and cannot report on subthreshold depolarizations. While voltagesensors could in principle be used to follow fast trains of action potentials and monitor subthresholddepolarizations, the low dynamic range of existing sensors makes them unsuitable for most experimentsin vivo. Moreover, there are presently no calcium or voltage sensors that are efficiently excited by red(550nm) wavelengths, a region of the spectrum that would enable their concurrent use with blue/greenabsorbinglight-activated channels.The goal of this proposal is to address these needs by developing a new generation of geneticallyencoded voltage biosensors with significantly improved dynamic range and speed and the ability to beused concurrently with light-activated channels. This work will enable finer and more powerful functionaldissection of the nervous system, while also establishing new protein engineering methods. The expertiseof the lab in fluorescent proteins engineering will be leveraged to educate future scientists and engineersabout research in protein engineering, and to introduce educators to the utility of genetically encodedbiosensors for teaching scientific concepts in laboratory courses.Intellectual MeritThe engineering objective of this proposal is to apply knowledge of fluorescence spectroscopyand protein structure to quantitatively improve the performance of an existing class of voltage sensors(Aim 1) and to develop an entirely new class of voltage sensors (Aim 2). In the first aim, we will increasedynamic range via rational optimization of Förster resonance energy transfer (FRET) between fluorescentproteins linked to a voltage sensing domain. In the second aim, we will use a conformationally sensitivered fluorescent protein to report on movements of a voltage sensing domain with rapid kinetics. For bothaims, we will employ rational design combined with comprehensive saturation mutagenesis and screeningof important sites.This project will be a pioneering study in molecular engineering in two ways. It will be the first torationally apply predictions from modeling of Förster resonance energy transfer to identify the factorslimiting the fluorescence output response, and then to perform protein engineering to comprehensivelyaddress those factors. Second, this project will be the first to combine comprehensive screening withatomic-level structural knowledge of sensing domains and fluorescent proteins in order to create anentirely new class of allosteric voltage sensors; this research will thus give insight on the relativeusefulness of prior knowledge and screening to the engineering of genetically encoded biosensors.Broader ImpactsVoltage sensors developed through this project will be promptly disseminated to the largerscientific community. These sensors will have a broad impact on the field of neuroscience by enablingrobust voltage sensing and concurrent control and readout of neuronal activity. Our work will also impactthe field of bioengineering by validating concepts and establishing strategies for how to optimize thedesign of fluorescent protein-based sensors using energy transfer or allostery.The proposed project will provide an excellent opportunity to introduce undergraduates and highschool students, including students from disadvantaged backgrounds, to research in molecularbioengineering. It will also provide the opportunity to introduce educators to the use of geneticallyencoded biosensing to create exciting laboratory lessons that teach important basic concepts whiledemonstrating the results of recent research in protein engineering.