Chromophore hydrolysis and release from photoactivated rhodopsin in native membranes.
Chromophore hydrolysis and release from photoactivated rhodopsin in native membranes.
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DOI：
10.1073/pnas.2213911119
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发表时间：
2022-11-08
影响因子：
11.1
通讯作者：
Palczewski, Krzysztof
中科院分区：
文献类型：
--
作者：
Hong, John D.;Salom, David;Kubas, Adam;Kiser, Philip D.;Palczewski, Krzysztof
当一个光子使视紫红质的11 - 顺式 - 视黄醛生色团异构化从而启动光转导时，视觉开始。全反式 - 视黄醛产物被水解，全反式 - 视黄醛释放出来，使得11 - 顺式 - 视黄醛能够重新结合以再生视紫红质从而维持视觉。随后，全反式 - 视黄醛被清除，通过还原为全反式 - 视黄醇来防止醛类毒性。这些代谢过程中的缺陷可能导致严重的视网膜病变。在其天然膜环境中研究负责视觉这些基本步骤的关键蛋白质的生物化学一直具有挑战性。利用快速定量化学和分析方法，我们直接捕获并量化了在天然膜中发生的这些基本生化过程的动力学和能量学。我们的结果阐明了整个视紫红质光循环和生色团再生，并且广泛适用于其他视黄醛蛋白。
为了维持视觉，光活化的视紫红质（Rho*）必须进行水解并释放全反式 - 视黄醛，为视觉循环产生底物以及可供11 - 顺式 - 视黄醛再生的脱辅基视蛋白。这种水解的动力学在视紫红质的天然膜环境中尚未被描述。我们开发了一种方法，包括通过异丙醇/硼氢化钠同时进行变性和生色团捕获，接着进行彻底的蛋白质消化、完全提取以及液相色谱 - 质谱分析。利用我们的方法，我们追踪了Rho*水解、随后与释放的全反式 - 视黄醛形成的N - 视黄醛 - 磷脂酰乙醇胺（N - ret - PE）加合物，以及全反式 - 视黄醛还原为全反式 - 视黄醇的过程。我们发现水解在天然膜中比在通常用于研究膜蛋白的去污剂胶束中发生得更快。在天然膜中水解的活化能被确定为17.7 ± 2.4千卡/摩尔。我们的数据支持这样的解释：变视紫红质II，即视紫红质的信号状态，是进行水解并释放其全反式 - 视黄醛的主要形式。在没有NADPH的情况下，游离的全反式 - 视黄醛与磷脂酰乙醇胺（PE）反应，形成大量的N - ret - PE（在生理pH下约占全反式 - 视黄醛总量的40%），其速率比Rho*水解快一个数量级。然而，N - ret - PE的形成因全反式 - 视黄醛被NADPH依赖的还原为全反式 - 视黄醇而大大减弱。N - ret - PE的形成和全反式 - 视黄醛的还原都不影响Rho*的水解速率。我们的研究提供了Rho*水解、全反式 - 视黄醛释放以及它重新进入视觉循环的全面情况，这个过程中的改变可能导致严重的视网膜病变。
Vision starts when a photon isomerizes the 11-cis-retinylidene chromophore of rhodopsin to initiate phototransduction. The all-trans-retinylidene product is hydrolyzed and all-trans-retinal released, allowing rebinding of 11-cis-retinal to regenerate rhodopsin for sustained vision. Subsequently, all-trans-retinal is cleared, preventing aldehyde toxicity by reduction to all-trans-retinol. Defects in these metabolic processes could lead to severe retinopathies. Studying the biochemistry of key proteins responsible for these fundamental steps of vision in their native membrane environments has remained challenging. Using rapid quantitative chemical and analytical methods, we directly captured and quantified the kinetics and energetics of these essential biochemical processes occurring in native membranes. Our results shed light on the entire rhodopsin photocycle and chromophore regeneration and are broadly applicable to other retinylidene proteins. For 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.
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影响因子：
3.3
作者：
Bartl, Franz J.;Vogel, Reiner
通讯作者：
Vogel, Reiner
DOI：
10.1016/s0022-5320(69)90005-7
发表时间：
1969-01-01
期刊：
JOURNAL OF ULTRASTRUCTURE RESEARCH
影响因子：
--
作者：
FALK, G;FATT, P
通讯作者：
FATT, P
影响因子：
4.4
作者：
Maeda, Akiko;Golczak, Marcin;Palczewski, Krzysztof
通讯作者：
Palczewski, Krzysztof
DOI：
10.1085/jgp.47.2.215
发表时间：
1963-11
期刊：
The Journal of general physiology
影响因子：
--
作者：
MATTHEWS RG;HUBBARD R;BROWN PK;WALD G
通讯作者：
WALD G
影响因子：
64.8
作者：
通讯作者：
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