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活性。到目前为止，光遗传学实验或由此产生的生物体疗法受到限制，因为激活光被周围组织强烈吸收。使用近红外（NIR）光是解决这个问题的一个潜在方案，但到目前为止，视紫红质对这个吸收区域不敏感。新视紫红质（NeoR），一种新的真菌视紫红质环化酶，我们最近发现的是能够吸收近红外光的第一次，因此作为一个模板打开这个光谱范围的其他视紫红质。除了其不寻常的近红外吸收，NeoR还具有高荧光和形成功能异二聚体的独特功能。除了NeoR之外，这些视紫红质-环化酶复合物还含有吸收蓝光或绿色光从而激活环化酶的常规视紫红质。在绿色光谱区具有灵敏度的同型二聚体视紫红质环化酶以前已被描述，而异源二聚体视紫红质复合物迄今为止是完全未知的。NeoR是一种光感受器，可以在NIR和UV吸收状态之间进行光切换，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在异源二聚体光感受器中的功能，解决以下问题：导致这些新型视紫红质环化酶仅作为异源二聚体发挥作用的结构特征是什么？NeoR亚基的光诱导状态如何影响环化酶活性？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质的不同之处。对于这些研究，我们正在使用不同的实验方法，如酶反应的动力学表征，以及使用静态和时间分辨光谱法阐明光诱导的结构变化。此外，我们希望研究来自相关真菌的同源蛋白，以鉴定适合于光遗传学应用的异二聚体视紫红质环化酶，并发现具有进一步红移吸收的NeoR变体。为了了解这些异二聚体光感受器的生理作用，特别是NeoR，我们打算研究能动真菌游动孢子的光取向，类似于同二聚体视紫红质环化酶的功能。NeoR中NIR活性发色团的结构和功能的详细知识将使光遗传学能够访问该光谱区域，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.