Cyanobacteriochromes (CBCRs) are photoswitchable linear tetrapyrrole (bilin)-based light sensors in the phytochrome superfamily with a broad spectral range from the near UV through the far red (330 to 760 nm). The recent discovery of far-red absorbing CBCRs (frCBCRs) has garnered considerable interest from the optogenetic and imaging communities because of the deep penetrance of far-red light into mammalian tissue and the small size of the CBCR protein scaffold. The present studies were undertaken to determine the structural basis for far-red absorption by JSC1_58120g3, a frCBCR from the thermophilic cyanobacterium Leptolyngbya sp. JSC-1 that is a representative member of a phylogenetically distinct class. Unlike most CBCRs that bind phycocyanobilin (PCB), a phycobilin naturally occurring in cyanobacteria and only a few eukaryotic phototrophs, JSC1_58120g3’s far-red absorption arises from incorporation of the PCB biosynthetic intermediate 18¹,18²-dihydrobiliverdin (18¹,18²-DHBV) rather than the more reduced and more abundant PCB. JSC1_58120g3 can also yield a far-red–absorbing adduct with the more widespread linear tetrapyrrole biliverdin IXα (BV), thus circumventing the need to coproduce or supplement optogenetic cell lines with PCB. Using high-resolution X-ray crystal structures of 18¹,18²-DHBV and BV adducts of JSC1_58120g3 along with structure-guided mutagenesis, we have defined residues critical for its verdin-binding preference and far-red absorption. Far-red sensing and verdin incorporation make this frCBCR lineage an attractive template for developing robust optogenetic and imaging reagents for deep tissue applications.

蓝细菌色素（CBCR）是光敏色素超家族中基于光开关线性四吡咯（bilin）的光传感器，其光谱范围从近紫外线到远红（330至760 nm）。远红外线吸收性CBCRs（frCBCRs）的最新发现引起了光遗传学和成像领域的极大兴趣，因为远红外线穿透哺乳动物组织的深度很大，而且CBCR蛋白支架的尺寸很小。进行了本研究以确定JSC1_58120g3（一种来自嗜热蓝细菌Leptolyngbya的frCBCR）吸收远红外光的结构基础。sp。JSC-1，是系统发育上独特的类的代表成员。不像大多数CBCRs了结合藻蓝胆素（PCB），一个藻胆素天然存在于蓝藻存在和只有几个真核光养，JSC1_58120g3的远红光吸收起因于PCB的生物合成中间体18的掺入¹，18 ² -dihydrobiliverdin（18 ¹，18 ² - DHBV），而不是减少更多，数量更多的PCB。JSC1_58120g3还可以与更广泛的线性四吡咯联运蛋白IXα（BV）产生吸收远红光的加合物，从而避免了与PCB共同生产或补充光遗传细胞系的需求。使用18的高分辨率X射线晶体结构¹，18 ²JSC1_58120g3的-DHBV和BV加合物以及结构指导的诱变作用，我们已经定义了对于其亲和素结合偏好和远红外吸收至关重要的残基。远红外感测和Verdin掺入使该frCBCR谱系成为吸引人的模板，可用于开发适用于深层组织的稳健的光遗传学和成像试剂。