Neorhodopsins, Near-infrared sensing Rhodopsin-cyclases

• Applicants: Professor Dr. Franz Bartl; Dr. Matthias Broser
• Subject Area: Biophysics ; Biochemistry ; Sensory and Behavioural Biology ; Biomaterials
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 509731234

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.

• DFG Programme: Research Grants