Chemical Biology of the Visual Pigments

摘要：视色素是人类视觉体验的起点，具有重要的生理意义， 并且也受到视网膜疾病的影响。因此，许多研究工作致力于 描述它们的结构-功能关系。尽管作出了这些努力， 了解视觉色素光化学和信号特性。对这一基本视觉的了解 生理学对于在开发相关视网膜病的治疗方面取得加速进展是必要的。在 所有视色素的核心是一种视黄醛发色团， 吸收适当波长的光子。这种复杂的反应，通过几个过程进行， 光中间体，触发光刺激传播到一个光刺激所必需的构象变化。 生化反应这种光活化过程以水解和释放视黄醇结束， 是受体光敏状态更新所必需的，因此是连续视觉功能所必需的。基本 关于视黄醇水解的受体结构、机制和调节剂仍存在问题 希夫碱以及小分子治疗候选物的相互作用模式。 在这里，我们将追求四个具体目标，这些目标采用了我们认为将 克服了以前无法克服的实验挑战。1)视紫红质结构解析 由纳米抗体稳定的光中间体。使用一系列新的骆驼抗体， 视紫红质光循环，我们将进行详细的结构-功能表征的变视紫红质 中间体的2)确定视紫红质和视锥细胞的视黄醛发色团的水解动力学 天然膜中的视蛋白色素。我们开发了一种新的基于质谱的方法， 首次直接检测天然膜中视色素的视网膜共轭状态;我们将 用这种方法来确定关键的速率常数必须模拟视觉色素之间的相互作用， 漂白周期和再生视觉周期。3)评估细胞溶质效应物和视觉 周期组分对基因敲除小鼠模型中视紫红质发色团水解速率的影响。使用 目的2中描述的方法，我们将表征Arr 1-/-，Grk 1-/-，Abca 4-/-， 和Rdh 8-/-小鼠，提供了新的见解，如何光和暗适应调制的 光转导和视觉周期蛋白。4)表征视紫红质的分子结构 与脂质和小分子的复合物。使用原生MS技术，我们 将量化与视紫红质在其各种激活状态下相关的磷脂。我们还将验证 体内小分子治疗候选物的药效学和药代动力学。我们相信 从这些研究中收集的信息将增强我们对视网膜疾病的分子水平的理解。 水平，并使其治疗的新策略的发展。

ABSTRACT: Visual pigments initiate the human visual experience, making them of great physiological interest, and also are affected in retinal diseases. Accordingly, numerous research efforts have been devoted to characterizing their structure-function relationships. Despite these efforts, critical gaps remain in our understanding of visual pigment photochemistry and signaling properties. Knowledge of this fundamental visual physiology is necessary to make accelerated progress in developing treatments for associated retinopathies. At the heart of all visual pigments is a retinaldehyde chromophore that undergoes a cis-trans isomerization upon absorption of a photon of a suitable wavelength. This complex reaction, which proceeds through several photointermediates, triggers the conformational changes necessary for the propagation of a light stimulus into a biochemical response. This photoactivation process ends with the hydrolysis and release of retinaldehyde, which is required for renewal of the receptor light-sensitive state and hence continuous visual function. Fundamental questions remain regarding receptor structure, mechanisms and modulators of hydrolysis of the retinaldehyde Schiff base, and the modes of interaction of small molecule therapeutic candidates. Here, we will pursue four specific aims that employ newly developed tools and approaches that we believe will overcome previously insurmountable experimental challenges. 1) Elucidate structures of rhodopsin photointermediates stabilized by nanobodies. Using a novel series of camelid antibodies that arrest the rhodopsin photocycle, we will perform a detailed structure-function characterization of metarhodopsin intermediates. 2) Define the kinetics of hydrolysis of the retinaldehyde chromophores of rhodopsin and cone opsin pigments in native membranes. We have developed a novel mass spectrometry-based method that can, for the first time, directly detect the retinal conjugation state of visual pigments in native membranes; we will use this method to determine key rate constants necessary to model the interplay between visual pigment bleaching cycles and the regenerative visual cycles. 3) Assess the influence of cytosolic effectors and visual cycle components on the rate of hydrolysis of rhodopsin chromophore in knockout mouse models. Using the methods described in Aim 2, we will characterize the rate of Schiff base hydrolysis in Arr1-/-, Grk1-/-, Abca4-/-, and Rdh8-/- mice, providing new insights into how light and dark adaptation are modulated by phototransduction and visual cycle proteins. 4) Characterize the molecular architecture of rhodopsin complexes with lipids and small molecules using native mass spectrometry. Using the native MS technique, we will quantify phospholipids that associate with rhodopsin in its various activation states. We will also validate the pharmacodynamics and pharmacokinetics of small molecule therapeutic candidates in vivo. We believe the information gleaned from these studies will enhance our understanding of retinal diseases at the molecular level and enable the development of novel strategies for their treatment.