Chromophore hydrolysis and release from photoactivated rhodopsin in native membranes

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dc.contributor.authorHong, John D.
dc.contributor.authorSalom, David
dc.contributor.authorKochman, Michał Andrzej
dc.contributor.authorKubas, Adam
dc.contributor.authorKiser, Philip
dc.contributor.authorPalczewski, Krzysztof
dc.contributor.organizationHerbert Eye Institute, Department of Ophthalmology, University of Californiaen
dc.contributor.organizationDepartment of Chemistry, University of Californiaen
dc.contributor.organizationInstitute of Physical Chemistry, Polish Academy of Sciencesen
dc.contributor.organizationDepartment of Physiology and Biophysics, University of Californiaen
dc.contributor.organizationDepartment of Clinical Pharmacy Practice, University of Californiaen
dc.contributor.organizationResearch Service, Veterans Affairs Long Beach Healthcare System, Long Beach, USAen
dc.contributor.organizationDepartment of Molecular Biology and Biochemistry, University of Californiaen
dc.date.accessioned2024-03-19T11:56:40Z
dc.date.available2024-03-19T11:56:40Z
dc.date.issued2022-11-02
dc.descriptionAAM
dc.description.abstractFor sustained vision, photoactivated rhodopsin (Rho*) must undergo hydrolysis and release of all-trans-retinal, producing substrate for the visual cycle and apo-opsin available for regeneration with 11-cis-retinal. The kinetics of this hydrolysis has yet to be described for rhodopsin in its native membrane environment. We developed a method consisting of simultaneous denaturation and chromophore trapping by isopropanol/borohydride, followed by exhaustive protein digestion, complete extraction, and liquid chromatography–mass spectrometry. Using our method, we tracked Rho* hydrolysis, the subsequent formation of N-retinylidene-phosphatidylethanolamine (N-ret-PE) adducts with the released all-trans-retinal, and the reduction of all-trans-retinal to all-trans-retinol. We found that hydrolysis occurred faster in native membranes than in detergent micelles typically used to study membrane proteins. The activation energy of the hydrolysis in native membranes was determined to be 17.7 ± 2.4 kcal/mol. Our data support the interpretation that metarhodopsin II, the signaling state of rhodopsin, is the primary species undergoing hydrolysis and release of its all-trans-retinal. In the absence of NADPH, free all-trans-retinal reacts with phosphatidylethanolamine (PE), forming a substantial amount of N-ret-PE (∼40% of total all-trans-retinal at physiological pH), at a rate that is an order of magnitude faster than Rho* hydrolysis. However, N-ret-PE formation was highly attenuated by NADPH-dependent reduction of all-trans-retinal to all-trans-retinol. Neither N-ret-PE formation nor all-trans-retinal reduction affected the rate of hydrolysis of Rho*. Our study provides a comprehensive picture of the hydrolysis of Rho* and the release of all-trans-retinal and its reentry into the visual cycle, a process in which alteration can lead to severe retinopathies.en
dc.description.sponsorship*work of AK funded by National Science Centre, Poland (Grant 2020/39/B/ST4/01952) *work of MAK funded by the program of the Polish Minister of Science and Higher Education entitled PMW in the years 2020 to 2024 (Agreement 5005/H2020-MSCA-COFUND/2019/2) *MAK acknowledges funding from the European Union Horizon 2020 research and innovation program under Marie Skłodowska-Curie Grant Agreement 847413 USA-related funds: We thank Dr. Gregory Tochtrop and Vladimir J. Kefalov and members of the P.D.K. and K.P. laboratories for helpful comments on this project. This research was supported in part by NIH Research Grant EY030873 (National Eye Institute) to K.P. and NIH Training Grants 1F30EY033659-01 and T32-GM08620 to J.D.H. The research was also supported by grants from the Department of Veterans Affairs (I01BX004939) and the NSF (CHE-2107713) to P.D.K. The authors acknowledge support to the Gavin Herbert Eye Institute at the University of California, Irvine from an unrestricted grant from Research to Prevent Blindness and from NIH core grant P30 EY034070.en
dc.identifier.citationPNAS 2022 Vol. 119 No. 45 e2213911119. https://doi.org/10.1073/pnas.2213911119en
dc.identifier.doi10.1073/pnas.2213911119
dc.identifier.issn0027-8424
dc.identifier.urihttps://open.icm.edu.pl/handle/123456789/24039
dc.language.isoen
dc.publisherNational Academy of Sciences USAen
dc.rightsUznanie autorstwa 4.0 Międzynarodowe*
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/*
dc.subjectrhodopsinen
dc.subjectchromophoreen
dc.subjectphotoreceptorsen
dc.subjectretinoidsen
dc.subjecteyeen
dc.titleChromophore hydrolysis and release from photoactivated rhodopsin in native membranesen
dc.typearticleen
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