In principle, highly ?uorescent rhodopsins could be employed as voltage sensors in neuroscience imaging. However, rhodopsin mutants featuring a ?uorescence ef?ciency close to the one of the green ?uorescence protein have not been discovered yet. Most importantly, we lack the molecular-level understanding required to predict if and how a speci?c set of mutations can amplify the weak ?uorescence of the rhodopsin chromophore. We propose to exploit state-of-the-art quantum chemical methods to systematically investigate these issues and to reengineer a microbial rhodopsin into a highly sensitive, genetically encodable, optogenetic tool for action potential visualization. -1-
In contrast to GFP and other cytoplasmic ?uorescent proteins, highly ?uorescent rhodopsins could be employed as membrane voltage sensors in neuroscience imaging to record the passage of an action potential using conventional light sources. If the ?uorescence is photo-switchable, the same rhodopsins could be employed in super-resolution microscopies to provide a spatiotemporal record of cell membrane processes not achievable using other probes. For these reasons we address the fundamental issue of the poor ?uorescence ef?ciency of natural or engineered rhodopsins which, even with laser sources, are at least 50 times less bright than green ?uorescent protein probes. To do so we develop novel computer models of a speci?c photochromic rhodopsin to address key mechanistic issues and open the way to the in silico search for bright mutants directly relevant to biomedical applications (e.g. the interrogation of neural circuits for studying how electrical signals contribute to higher order functions and to their dysfunctional states). -1-
| Manathunga, Madushanka; Yang, Xuchun; Olivucci, Massimo (2018) Electronic State Mixing Controls the Photoreactivity of a Rhodopsin with all- trans Chromophore Analogues. J Phys Chem Lett 9:6350-6355 |
