Neorhodopsins, Near-infrared sensing Rhodopsin-cyclases

微生物视紫红质是光遗传学中的关键蛋白质，因为它们能够通过外部光控制细胞的活动。到目前为止，在活体上进行的光遗传实验或由此产生的疗法一直受到限制，因为激活的光被周围组织强烈吸收。近红外光的使用是这个问题的潜在解决方案，但到目前为止，视紫红质对这个吸收区域不敏感。新视紫质(NeoR)是我们最近发现的一种新的真菌视紫红质环化酶，它首次能够吸收近红外光，从而为其他视紫红质开辟了这一光谱范围。除了其不同寻常的近红外吸收外，NeoR还具有高荧光和形成功能异二聚体的独特特征。除了NeoR，这些视紫红质-环化酶复合体还含有一种传统的视紫红质，可以吸收蓝光或绿光，从而激活环化酶。在绿色光谱区域敏感的同二聚体视紫红质环化酶已被描述，而异二聚体视紫红质复合体到目前为止还完全未知。NeoR是一种双稳态的光感受器，可以在近红外和紫外吸收状态之间切换，从而以一种未知的方式影响酶的活性。该项目的目的是阐明NeoR在异二聚体感光细胞中的功能，解决以下问题：导致这些新型视紫红质环化酶仅作为异二聚体发挥功能的结构特征是什么？NeoR亚单位的光诱导状态如何影响环化酶的活性？此外，我们的目标是研究NeoR的光化学，以了解它与传统视紫红素的不同之处。对于这些研究，我们使用不同的实验方法，例如酶反应的动力学表征，以及使用静态和时间分辨光谱来阐明光诱导的结构变化。此外，我们想要研究相关真菌的同源蛋白质，以鉴定适合于光遗传应用的异二聚体视紫红质环化酶，并寻找具有进一步红移吸收的NeoR变体。为了了解这些异二聚体感光器的生理作用，特别是NeoR，我们打算研究游动真菌游动孢子的光定位，类似于同源二聚体视紫红质环化酶的功能。对NeoR中近红外活性发色团的结构和功能的详细了解将使光遗传学能够访问该光谱区域的策略成为可能，这将允许全新的应用。

Microbial rhodopsins are key proteins in optogenetics, since theyenable the control of cellular activity by external light. Until now, optogenetic experiments or resulting therapies in living organismshave been limited because the activating light is strongly absorbed by the surrounding tissue. The use of near-infrared (NIR) light is a potential solution for this problem, but so far rhodopsins were not sensitive for this absorption region. Neorhodopsin (NeoR), a novel fungal Rhodopsin-cyclase that we recently discovered is capable of absorbing NIR light for the first time and therefore serves as a template for opening this spectral range for other rhodopsins. In addition to its unusual NIR absorption, NeoR has the unique features of being highly fluorescent and forming functional heterodimers. Besides NeoR, these rhodopsin-cyclase complexes contain a conventional rhodopsin that absorbs blue or green light and thereby activates the cyclase. Homodimeric Rhodopsin-cyclases with sensitivity in the green spectral region have been previouslydescribed, whereas heterodimeric rhodopsin complexes are hitherto completely unknown. NeoR is a bistable photoreceptor and is photoswitchable between a NIR and a UV absorbing state, thus affecting the enzyme activity in a yet unknown way. The aim of the proposed project is to elucidate the functioning of NeoR in the heterodimeric photoreceptor addressing the following questions: What are the structural features that cause these novel Rhodopsin-cyclases to function only as heterodimers? How does the light-induced state of the NeoR subunit affect the cyclase activity? In addition, we aim to study the photochemistry of NeoR to understand how it differs from conventional rhodopsins. For these studies, we are using different experimental approaches, such as the kinetic characterization of the enzyme reaction, as well as the elucidation of light-induced structural changes using static and time-resolved spectroscopy. Furthermore, we want to investigate homologous proteins from related fungi in order to identify heterodimeric Rhodopsin-cyclase suitable for optogenetic applications and to find NeoR variants with further red-shifted absorption. To understand the physiological role of these heterodimeric photoreceptors and in particular of NeoR, we intend to study the photo-orientation of motile fungal zoospores, in analogy to function of homodimeric Rhodopsin-cyclase. Detailed knowledge of the structure and function of the NIR-active chromophore in NeoR will enable strategies for making this spectral region accessible to optogenetics, which would allow completely new applications.