Structural investigations of native and mutant human green cone pigment

描述（由申请人提供）：绿色视锥色素在530 nm波长处吸收最大光。视网膜中的三种锥状色素具有不同的光吸收光谱，使三色色觉成为可能。尽管具有独特的吸收光谱，但这三种色素利用相同的化学发色团11-顺式-视黄醛。光的吸收将11-顺式-视黄醛异构化为全反式-视黄醛，并激活色素蛋白以将信号向下传播视觉级联。了解绿色视锥色素如何特别影响发色团以最大限度地吸收530 nm波长的光，将揭示视觉功能的基本机制。这将通过X射线晶体学结合详细的量子化学计算确定高分辨率蛋白质结构来实现。结合实验和理论揭示的结构构象和化学相互作用将证明蛋白质对生色团的影响。有了这些新的见解，两个突变将被引入和表征其对绿色锥色素的结构的影响。W177 R绿色色素突变阻止蛋白质正确折叠，导致蛋白质保留在内质网中。将使用Rosetta蛋白质结构预测套件研究这种总体结构扰动。能量计算将揭示W177 R突变中缺乏的关键相互作用，这些相互作用是正确的蛋白质折叠所必需的。A180 S突变不改变绿色视锥色素的折叠。为了了解A180 S突变改变吸光度的机制，我们将通过X射线晶体学实验确定突变色素的结构。与天然绿色视锥色素一样，实验和理论的结合将用于理解A180 S突变的结构和化学效应。锥状色素的致病突变很少，这是开发人类色觉缺陷潜在治疗方法的关键步骤。

DESCRIPTION (provided by applicant): The green cone pigment absorbs light maximally at a wavelength of 530 nm. Three cone pigments in the retina with distinct light absorbance spectra make trichromatic color vision possible. Despite having unique absorbance spectra, these three pigments utilize the same chemical chromophore, 11-cis-retinal. Absorbance of light isomerizes 11-cis-retinal to all-trans-retinal and activates the pigment protein to propagate the signal down the visual cascade. Understanding how green cone pigment specifically affects the chromophore to maximally absorb 530 nm wavelength light will reveal a basic mechanism of visual function. This will be accomplished by determining a high resolution protein structure by X-ray crystallography together with detailed quantum chemical calculations. The structural conformation and chemical interactions revealed by combining experiments with theory will demonstrate the influence of the protein on the chromophore. With these new insights, two mutations will be introduced and characterized for their influence on the structure of the green cone pigment. The W177R green pigment mutation prevents the protein from folding properly, causing the protein to be retained in the endoplasmic reticulum. This gross structural perturbation will be investigated using the Rosetta protein structure prediction suite. Energetic calculations will reveal key interactions lacking in the W177R mutation that are required for proper protein folding. The A180S mutation does not alter folding of the green cone pigment. To understand the mechanism by which the A180S mutation alters absorbance, we will determine the structure of the mutant pigment experimentally by X-ray crystallography. As with native green cone pigment, a combination of experiment and theory will be used to understand the structural and chemical effects of the A180S mutation. Very few pathogenic mutations have been characterized for cone pigments, a critical step in developing potential therapeutics for deficiencies in human color vision.