Opsineering: Engineering Novel ChannelRhodospins for Optogenetics Applications

Opsineering：为光遗传学应用设计新型通道Rhodospins

• 批准号: 9125904
• 负责人: Claire Nicole Bedbrook
• 金额: $ 3.72万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9125904
• 关键词: Address; Animals; Autistic Disorder; Biological; Biological Assay; Brain; Brain region; Cations; Cells; Characteristics; Chemical Stimulation; Chimera organism; Collaborations; Color; Development; Diagnosis; Directed Molecular Evolution; Drug abuse; Electric Stimulation; Electrophysiology (science); Engineering; Evolution; Family; Foundations; Gated Ion Channel; Genetic Recombination; Goals; Health; Homologous Gene; Ions; Kinetics; Laboratories; Length; Libraries; Life; Light; Literature; Mammalian Cell; Membrane; Mental Depression; Mental disorders; Methods; Modeling; Mutate; Mutation; Neurobiology; Neurons; Neurosciences; Opsin; Organism; Parkinson Disease; Pathway interactions; Permeability; Photophobia; Population Heterogeneity; Probability; Property; Protein Engineering; Proteins; Research; Research Training; Schizophrenia; Specific qualifier value; Specificity; Speed; Stimulus; Structure; System; Techniques; Time; Transgenic Organisms; Variant; Work; addiction; base; brain circuitry; cell type; desensitization; design; improved; light gated; member; microbial; millisecond; mutant; nervous system disorder; neuronal circuitry; next generation; novel; optogenetics; protein function; screening; single molecule; success; tool

DESCRIPTION (provided by applicant): Optogenetics is genetically encoded, optically induced, control of cells through transgenic expression of microbial opsins in mammalian neurons. When these opsins are expressed in a cell-type specific manner and light activated, they provide temporally and spatially separated stimulation of independent hyperpolarizing and depolarizing channels in neurons in living animals. Channelrhodopsins (ChRs) are the microbial opsins used in optogenetics to trigger light induced depolarization. ChRs are light-gated ion channels that operate on the order of milliseconds, a time scale relevant for neuronal activation, and can be expressed in the membrane of distinct cell types with high temporal precision in well-defined brain regions. This contrasts with the poor temporal dynamics or lack of specificity of chemical or electrical stimulation methods. However, the optogenetics tools currently available for neuronal circuit interrogation are limited based on expression, light-wavelength activation, kinetics and ion specificity. Our proposed project addresses these limitations through protein engineering. Protein engineering through directed evolution and structure-guided recombination are well-established methods for modifying and optimizing proteins for desired functions. Current literature and preliminary collaborative work between the Gradinaru and Arnold labs at Caltech indicate that channelrhodopsins are amenable to functionally useful laboratory evolution and manipulation. This work will be focused toward engineering improved channelrhodopsins for use as biological tools in optogenetics. The aim is to engineer channelrhodopsins for optimal ion selectivity, kinetics, reversibility, and shifted light excitatio wavelengths. These new channel proteins will have applications in probing the brain's circuitry to better understand and model healthy and non-healthy brain function as a foundation for controlling and diagnosing neurological disorders such as addiction, depression and Parkinson's disease.

描述(由申请人提供)：光遗传学是通过在哺乳动物神经元中转基因表达微生物视蛋白来遗传编码、光诱导、控制细胞的。当这些Opsins以细胞类型特异性的方式表达并被光激活时，它们为活着的动物神经元中独立的超极化和去极化通道提供时空分离的刺激。通道视紫红质(CHRS)是光遗传学中用于触发光诱导去偏振的微生物视黄素。CHRs是一种光门离子通道，工作在毫秒量级，这是一个与神经元激活相关的时间尺度，可以在定义明确的大脑区域以高时间精度在不同类型的细胞膜上表达。这与化学或电刺激方法的时间动力学差或缺乏特异性形成了鲜明对比。然而，目前可用于神经元电路询问的光遗传学工具基于表达、光波长激活、动力学和离子特异性而受到限制。我们提出的项目通过蛋白质工程解决了这些限制。通过定向进化和结构导向重组进行蛋白质工程是修饰和优化蛋白质以实现所需功能的成熟方法。目前的文献和加州理工大学Gradinaru和Arnold实验室之间的初步合作工作表明，通道视紫红质服从于功能有用的实验室进化和操纵。这项工作将集中在设计改进的通道视紫红质作为光遗传学中的生物工具。其目的是设计通道视紫红质，以获得最佳的离子选择性、动力学、可逆性和移动的光激发波长。这些新的通道蛋白将应用于探测大脑的电路，以更好地了解和模拟健康和非健康的大脑功能，作为控制和诊断成瘾、抑郁和帕金森氏症等神经疾病的基础。

项目成果

Machine learning to design integral membrane channelrhodopsins for efficient eukaryotic expression and plasma membrane localization.

• DOI: 10.1371/journal.pcbi.1005786
• 发表时间: 2017-10
• 期刊: PLoS computational biology
• 影响因子: 4.3
• 作者: Bedbrook CN;Yang KK;Rice AJ;Gradinaru V;Arnold FH
• 通讯作者: Arnold FH

Recent advances in engineering microbial rhodopsins for optogenetics.

用于光遗传学的工程微生物动蛋白的最新进展。

• DOI: 10.1016/j.sbi.2015.05.001
• 发表时间: 2015-08
• 期刊: Current opinion in structural biology
• 影响因子: 6.8
• 作者: McIsaac RS;Bedbrook CN;Arnold FH
• 通讯作者: Arnold FH

Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore.

• DOI: 10.1016/j.chembiol.2017.02.008
• 发表时间: 2017-03-16
• 期刊: Cell chemical biology
• 影响因子: 8.6
• 作者: Herwig L;Rice AJ;Bedbrook CN;Zhang RK;Lignell A;Cahn JKB;Renata H;Dodani SC;Cho I;Cai L;Gradinaru V;Arnold FH
• 通讯作者: Arnold FH