Atomistic design of microbial opsin-based blue-shifted optogenetics tools

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.

The light‐driven sodium ion pump: A new player in rhodopsin research

“…In addition, as shown in recent successful examples [20,33,[69][70][71][72][73][74][75][76][77][78], the wealth of structural data available and advances in genomic technologies will lead to further engineering and discovery of functionally novel rhodopsins, and it is expected that the scope and impact of rhodopsin research will continue to expand.…”

“…Recently, the structural information on channelrhodopsin (ChR), a light-gated cation channel, has prompted the engineering of a light-gated anion channel [69][70][71][72], and soon after that, light-gated anion channels were discovered in a natural source [73,74]. Structurebased molecular engineering accomplished a 100 nm blue shift in the absorption spectrum of a proton pumping rhodopsin [75], and de novo transcriptome sequencing of green alga has led to the discovery of a novel ChR with a very red-shifted spectral peak (590 nm) [76]. Likewise, structural information on KR2 enabled us to design a lightdriven outward K þ pump (KR2 Kþ ) [33]: though the functionality in nature still not known, it is highly likely that we will be able to identify one in the near future.…”

Molecular Mechanism of Wide Photoabsorption Spectral Shifts of Color Variants of Human Cellular Retinol Binding Protein II

“…The other contributions originate from torsion around C 6 −C 7 bond. 4 The torsion by 154°in M4 is relaxed to be ∼168°in M10 and M11, giving contributions of ∼0.5 kcal/mol to the red shifts ( Figure S17).…”

“…Molecular mechanism of the color regulation of the opsin shift has extensively been investigated to obtain a molecular understanding of color vision as well as guiding principles for engineering of color variants of microbial-opsin based optogenetics tools to augment controllability of neuronal activity. 3,4 Recently, Wang et al engineered a soluble protein, human cellular retinol binding protein II (hCRBPII), and created mutants that can bind retinal with PSB linkage through Q108K mutation ( Figure 1) and exhibit anomalously large spectral shifts. 5 Absorption maxima of the color variants made by point mutations at 10 positions at most widely range over ∼200 nm from 425 to 644 nm.…”

The form and function of channelrhodopsin

“…Absorption peaks for known ChRs span 440 to 590 nm (1, 41, 50, 65), and 630- to 644-nm peaks are seen in certain other retinal-binding proteins (66, 67). Absorption spectra are determined by factors including RBP polarity, ATR planarity [in particular, coplanarity of the β-ionone ring C6=C7 bond with the polyene chain in the 6-s conformation (68)], and connection of negatively charged RSBH counterions with long-range hydrogen-bonding networks (68–70) (Figs. 1 and 4).…”