New Fluorescent Probes for Visual Proteins. Part III. Probing Auxiliary Gateways and the Stepwise Activation Model of Ligand Transport.
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| Title: | New Fluorescent Probes for Visual Proteins. Part III. Probing Auxiliary Gateways and the Stepwise Activation Model of Ligand Transport. |
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| Authors: | Papper, Vladislav1 (AUTHOR) vpapper@addu.edu.ph |
| Source: | Journal of Fluorescence. Jun2026, Vol. 36 Issue 6, p3753-3775. 23p. |
| Subjects: | Visual pigments, Fluorescent probes, Ligand binding (Biochemistry), Opsins, Rhodopsin |
| Abstract: | This study presents the functional characterisation of auxiliary ligand-transport pathways in bovine visual pigments using the fluorescent probe L1, a synthetic analogue of retinal. While structural studies have identified potential entrance channels Hole A and Hole B, the dynamic 'traffic report' of how these pathways regulate retinoid flux has remained elusive. We utilised the unique photophysical properties of L1, specifically its dual-fluorescence and sensitive response to the local dielectric environment, to probe the subsurface reactive gateways of opsin and rhodopsin. In the rigid, hydrophobic environment of the protein channels, the L1 probe exhibits a significant increase in fluorescence quantum yield, reaching in bovine opsin, which represents a significant enhancement relative to the aqueous state. This enhancement is a direct reporter of the mechanical planarisation of the probe, which effectively blocks the non-radiative twisted intramolecular charge-transfer (TICT) state prevalent in fluid solvents. Competitive titration and time-resolved kinetic analysis identify a critical stoichiometric transition in the ligand channelling mechanism. Our results demonstrate that the auxiliary binding sites function as a high-capacity molecular buffer, accommodating up to seven equivalents of the L1 probe without impeding the primary visual cycle. However, exceeding this threshold leads to a distinct 'saturation cliff', where the auxiliary channels become saturated and act as kinetic bottlenecks. At 20 equivalents of L1, the recovery efficiency of native rhodopsin is suppressed to only 10.5%, providing the functional evidence for the gated nature of these pathways. Reciprocal inhibition experiments confirm that L1 and 11-cis-retinal utilise a shared network of subsurface reactive gateways, while the rigid, linear all-trans-retinal and the shorter L2 probe remain excluded due to geometrical selectivity. These findings culminate in the proposal of a stepwise activation model, where the auxiliary sites act as a molecular reservoir that regulates the flux of retinoids into the primary orthosteric pocket. This model provides a robust physical foundation for the memory vision hypothesis, suggesting that the occupancy state of these channels governs the persistence of visual signals following intense light exposure. By contrasting the 'open/gated' architecture of the bovine visual pigments with the 'sealed' and rigid structure of bacteriorhodopsin, we highlight the evolutionary specialisation required for high-efficiency vertebrate vision. [ABSTRACT FROM AUTHOR] |
| Copyright of Journal of Fluorescence is the property of Springer Nature and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) | |
| Database: | Engineering Source |
| FullText | Text: Availability: 0 |
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| Header | DbId: egs DbLabel: Engineering Source An: 195093920 AccessLevel: 6 PubType: Academic Journal PubTypeId: academicJournal PreciseRelevancyScore: 0 |
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| Items | – Name: Title Label: Title Group: Ti Data: New Fluorescent Probes for Visual Proteins. Part III. Probing Auxiliary Gateways and the Stepwise Activation Model of Ligand Transport. – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AR" term="%22Papper%2C+Vladislav%22">Papper, Vladislav</searchLink><relatesTo>1</relatesTo> (AUTHOR)<i> vpapper@addu.edu.ph</i> – Name: TitleSource Label: Source Group: Src Data: <searchLink fieldCode="JN" term="%22Journal+of+Fluorescence%22">Journal of Fluorescence</searchLink>. Jun2026, Vol. 36 Issue 6, p3753-3775. 23p. – Name: Subject Label: Subjects Group: Su Data: <searchLink fieldCode="DE" term="%22Visual+pigments%22">Visual pigments</searchLink><br /><searchLink fieldCode="DE" term="%22Fluorescent+probes%22">Fluorescent probes</searchLink><br /><searchLink fieldCode="DE" term="%22Ligand+binding+%28Biochemistry%29%22">Ligand binding (Biochemistry)</searchLink><br /><searchLink fieldCode="DE" term="%22Opsins%22">Opsins</searchLink><br /><searchLink fieldCode="DE" term="%22Rhodopsin%22">Rhodopsin</searchLink> – Name: Abstract Label: Abstract Group: Ab Data: This study presents the functional characterisation of auxiliary ligand-transport pathways in bovine visual pigments using the fluorescent probe L1, a synthetic analogue of retinal. While structural studies have identified potential entrance channels Hole A and Hole B, the dynamic 'traffic report' of how these pathways regulate retinoid flux has remained elusive. We utilised the unique photophysical properties of L1, specifically its dual-fluorescence and sensitive response to the local dielectric environment, to probe the subsurface reactive gateways of opsin and rhodopsin. In the rigid, hydrophobic environment of the protein channels, the L1 probe exhibits a significant increase in fluorescence quantum yield, reaching in bovine opsin, which represents a significant enhancement relative to the aqueous state. This enhancement is a direct reporter of the mechanical planarisation of the probe, which effectively blocks the non-radiative twisted intramolecular charge-transfer (TICT) state prevalent in fluid solvents. Competitive titration and time-resolved kinetic analysis identify a critical stoichiometric transition in the ligand channelling mechanism. Our results demonstrate that the auxiliary binding sites function as a high-capacity molecular buffer, accommodating up to seven equivalents of the L1 probe without impeding the primary visual cycle. However, exceeding this threshold leads to a distinct 'saturation cliff', where the auxiliary channels become saturated and act as kinetic bottlenecks. At 20 equivalents of L1, the recovery efficiency of native rhodopsin is suppressed to only 10.5%, providing the functional evidence for the gated nature of these pathways. Reciprocal inhibition experiments confirm that L1 and 11-cis-retinal utilise a shared network of subsurface reactive gateways, while the rigid, linear all-trans-retinal and the shorter L2 probe remain excluded due to geometrical selectivity. These findings culminate in the proposal of a stepwise activation model, where the auxiliary sites act as a molecular reservoir that regulates the flux of retinoids into the primary orthosteric pocket. This model provides a robust physical foundation for the memory vision hypothesis, suggesting that the occupancy state of these channels governs the persistence of visual signals following intense light exposure. By contrasting the 'open/gated' architecture of the bovine visual pigments with the 'sealed' and rigid structure of bacteriorhodopsin, we highlight the evolutionary specialisation required for high-efficiency vertebrate vision. [ABSTRACT FROM AUTHOR] – Name: AbstractSuppliedCopyright Label: Group: Ab Data: <i>Copyright of Journal of Fluorescence is the property of Springer Nature and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.) |
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| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.1007/s10895-026-04802-8 Languages: – Code: eng Text: English PhysicalDescription: Pagination: PageCount: 23 StartPage: 3753 Subjects: – SubjectFull: Visual pigments Type: general – SubjectFull: Fluorescent probes Type: general – SubjectFull: Ligand binding (Biochemistry) Type: general – SubjectFull: Opsins Type: general – SubjectFull: Rhodopsin Type: general Titles: – TitleFull: New Fluorescent Probes for Visual Proteins. Part III. Probing Auxiliary Gateways and the Stepwise Activation Model of Ligand Transport. Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: Papper, Vladislav IsPartOfRelationships: – BibEntity: Dates: – D: 01 M: 06 Text: Jun2026 Type: published Y: 2026 Identifiers: – Type: issn-print Value: 10530509 Numbering: – Type: volume Value: 36 – Type: issue Value: 6 Titles: – TitleFull: Journal of Fluorescence Type: main |
| ResultId | 1 |
