Engineering of Chrimson for Subcellular Optogenetic application

该项目的目的是进一步表征和工程化Chrimson，具有最红移吸收光谱和高质子选择性的通道视紫红质，其与古细菌光驱动质子泵和大多数其他通道视紫红质都具有结构特征。我们将利用生物化学、生物物理和理论方法，重点研究包括活性位点、外门和选择性过滤器在内的不寻常的亚结构。为了彻底理解，我们将建立和研究计算模型，这将有助于解释实验结果，并有助于合理设计红移变体。我们将设计新的甚至更多的红移变体，增加钠选择性和/或修改动力学。由于在超过600 nm的波长下神经元组织的传输增加，这些Chrimson衍生物对于神经科学中的体内应用是最感兴趣的。此外，我们将探索生物变色特性，以消除或促进Chrimson的双峰切换。我们将为隔室光遗传学定制具有长开放状态和高质子选择性的新Chrimson变体，特别是用于耗尽突触囊泡和溶酶体中的质子，获得对其酸化和功能化的光遗传学控制。

The aim of the project is to further characterize and engineer Chrimson, the channel-rhodopsin with the most red shifted absorption spectrum and high proton selectivity, which shares structural features both with archaeal light driven proton pumps and most other channelrhodopsin. We will focus our studies on the unusual substructures including the active site, the outer gate, and the selectivity filter by using biochemical, biophysical and theoretical approaches. For a thorough understanding, we will build and investigate computational models, which will help to interpret experimental findings and assist in the rational design of red-shifted variants. We will engineer new and even more red-shifted variants with increased sodium-selectivity and/or modified kinetics. Due to the increased transmission of neuronal tissues at wavelength beyond 600 nm, these Chrimson derivatives are of outmost interest for in vivo applications in neuroscience. Furthermore, we will explore the biochromic properties to either eliminate or facilitate bimodal switching of Chrimson. We will tailor new Chrimson variants with long open states and high proton selectivity for compartmental optogenetics, specifically for depleting protons from synaptic vesicle and lysosome, gaining optogenetic control of their acidification and functionalization.