Engineering of Chrimson for Subcellular Optogenetic application

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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.