Abstract
Microbial opsins with a bound chromophore function as photosensitive ion transporters and have been employed in optogenetics for the optical control of neuronal activity. Molecular engineering has been utilized to create colour variants for the functional augmentation of optogenetics tools, but was limited by the complexity of the protein–chromophore interactions. Here we report the development of blue-shifted colour variants by rational design at atomic resolution, achieved through accurate hybrid molecular simulations, electrophysiology and X-ray crystallography. The molecular simulation models and the crystal structure reveal the precisely designed conformational changes of the chromophore induced by combinatory mutations that shrink its π-conjugated system which, together with electrostatic tuning, produce large blue shifts of the absorption spectra by maximally 100 nm, while maintaining photosensitive ion transport activities. The design principle we elaborate is applicable to other microbial opsins, and clarifies the underlying molecular mechanism of the blue-shifted action spectra of microbial opsins recently isolated from natural sources.
Highlights
Microbial opsins with a bound chromophore function as photosensitive ion transporters and have been employed in optogenetics for the optical control of neuronal activity
Similar to the fluorescent proteins utilized in cell visualization, the functionalities of the microbial opsins in optogenetics have been diversified through genomic searches for analogous light-sensitive ion transporters and molecular engineering[10]
In the native proteins of C1C2 and AR3, as well as those of microbial rhodopsins with presently available X-ray crystallographic structures, the methyl groups at positions 1 and 5 (C16, C17, and C18 methyl groups; see Fig. 1a) in the b-ionone ring of the chromophore are recognized by cavities created by the well-conserved residues of the binding pocket, which maintain the orientation of the ring alignment in the same plane as the polyene chain (Fig. 1b–g, Supplementary Fig 1 and Supplementary Table 1)
Summary
Microbial opsins with a bound chromophore function as photosensitive ion transporters and have been employed in optogenetics for the optical control of neuronal activity. Point mutations at 13 positions introduced in phoborhodopsin (lmax 1⁄4 497 nm), guided based on the X-ray crystallographic structure[19,20,21,22] to make the sequential and spatial alignments of the mutated residues in the binding pocket identical to those of a largely red-shifted microbial opsin, bacteriorhodopsin from Halobacterium salinarum (HsBR, lmax 1⁄4 568 nm), a light-driven proton pump, shifted the absorption maximum by 30 nm, which is only 40% of the overall 70 nm spectral shift of the native proteins[23] This observation indicated that the creation of colour variants requires precise structural design at atomic resolution based on physicochemical considerations. The present study demonstrates that highly resolved atomistic design provides a strong basis for the production of colour variants through minimal modification that is universally applicable to analogous proteins
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
