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  1. Evaluation Summary:

    This manuscript describes an investigation of the evolution of monostable rhodopsins, typically found in vertebrates. It highlights that single amino acid changes in vertebrate rhodopsins can create a partial bistable retinal pigment that can be photoconverted back to the ground state or it will slowly convert back to the ground state retinal isomer. The rationale for the experiments came from the discovery of a very interesting activation mechanism of the nonvisual pigment Opn5L1. This work has important implications for how our visual pigments have been optimized during evolution, and it contributes important insights into engineering bistable pigments for optogenetic applications.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    The manuscript addresses the important question about the inner mechanics that makes photoreactions of vertebrate rhodopsins non-cyclic and include the release of the isomerized retinal whereas invertebrate rhodopsins undergo cyclic reactions or may switch forward and backward by light ( bistable rhodopsins).

    Experiments are well done and convincing, clearly documented and presented by the figures.

    The discussion of the interpretation why G188C mutant prevents retinal release during the Meta II state and its thermal or photochemical anti/syn isomerization could be done much better. Many invertebrate rhodopsins do not release the retinal during the Meta II state and do not have any Cys residue at this location. Moreover, some of them deprotonate during the meta state and others do not. The authors are experts in invertebrate rhodopsins as well and privileged to give a better interpretations.

    Next, the suggestion that Cys causes transient thioadduct formation during the photocycle is not justified by anything and should be done with more care. For example a Cys at a similar position has been found in microbial Channelrhodopsins (ChRs) and has been shown to be critical for the photocycle kinetics and for anti/syn isomerization. The sulfur of the Cys seems to act as a nucleophile for retinal polyene chain, forming a thioadduct as in OPN5L but not in other rhodopsin. The Cys obviously influences the charge distribution in darkness and during the excited state. Moreover, residues in the active site including E113, E188 and other residues could act as steric constrains that influence the isomerization specificity as well.

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  3. Reviewer #2 (Public Review):

    The studies of the non-visual pigment Opn5L1, that preceded this study, suggested a very interesting mechanism and it indicated bistability of the pigment. Starting from these insights, the authors attempted successfully to transfer key properties of Opn5L1 to rhodopsin. Their data clearly indicates that a mutation of a glycine residue at position 188 of rhodopsin to a cysteine residue, which is well conserved in Opn5L1 related photo pigments, makes the retinal protein photo cyclic and photo reversible. This indicates that residue 188 contributes to the diversification of photoreactions in opsins. The spectroscopic data in combination with signalling assays and the determination of retinal isomers fully support the claims of the authors. The photo reversibility is shown by using UV light illumination that is clearly increased in the mutated protein over the wild type protein. In addition, the slow thermal reversion can be unambiguously derived from the provided data. To achieve these experimental results, it was essential to use a stabilization strategy of the opsins. Similar strategies have been previously used to study retinal uptake in retinitis pigmentosa mutations and rhodopsin structures successfully. Stabilization is introduced by the double mutation N2C/D282C which crosslinks the extracellular N terminal domain to the receptor. It is known that this modification does not result in significant thermos stabilisation and no interference with spectroscopic properties and light activation. The presented work really proofs that the equivalent residue in Opn5L1 to residue 188 in rhodopsin is very important for the bistable nature of this retinal pigment. The study highlights a very important link between invertebrate bistable pigments and our vertebrate visual pigment and it makes it likely that our low light sensitive optimized GPCRs have evolved from the more ancient invertebrate-like bistable pigments. This is an outstanding scientific achievement for our understanding of the visual pigments. The study also has implications for our understanding of bistable pigments, and for the engineering of retinal proteins for optogenetic applications. The work is very well executed and the data is justifying the scientific conclusions.

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  4. Reviewer #3 (Public Review):

    The authors explored the role of residue 188 (Gly in vertebrate opsins) in the functional properties of photopigment (monostable vs bistable). Their focus on 188Cys mutant is based on the fact that in photocyclic Opn5L1 this position is occupied by a cysteine. They showed that rhodopsin with Cys at position 188 can convert all-trans retinal (active state) into 11- or -9-cis (dark state), in contrast to wild-type rhodopsin. They also show that G protein activation by Cys188 rhodopsin is not as prolonged as by wild type.

    While the authors' experiments address an important biological issue, the manuscript does not state this explicitly: what is the advantage of monostable vertebrate rhodopsin over bistable invertebrate one? After all, vertebrates paid a very high price for this: it necessitated specialized mechanisms of delivery of all-trans-retinal from rods to RPE and 11-cys form from RPE to rods, as well as a multi-enzyme visual cycle that converts all-trans-retinal to 11-cis. The only plausible explanation proposed so far is that monostable rhodopsin undergoes a greater conformational change upon light absorption, and therefore activates many more transducin molecules per photoisomerization. In fact, the authors' data support this notion. This increases the light sensitivity of the system. It was established that vertebrate rods sense single photons, whereas invertebrate photoreceptors do not.

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