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首页> 外文期刊>Biochemistry >Characterization of Membrane Protein Non-native States. 2. The SDS-UnfoldedStates of Rhodopsint
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Characterization of Membrane Protein Non-native States. 2. The SDS-UnfoldedStates of Rhodopsint

机译：膜蛋白非天然状态的表征。 2.红景天的SDS-未折叠状态

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Little is known about the molecular nature of residual structure in unfolded states of membrane proteins. A screen of chemical denaturants to maximally unfold the mammalian membrane protein and prototypic G protein coupled receptor rhodopsin, without interference from aggregation, described in an accompanying paper (DOI 0.102 I /bil00338e), identified sodium dodecyl sulfate (SDS), alone or in combination with other chemicals, as the most suitable denaturant. Here, we initiate the biophysical characterization of SDS-denatured states of rhodopsin. Using absorption, steady-state and time-resolved fluorescence spectroscopy, dynamic light scattering, and cysteine accessibility studies, tertiary structure of denatured states was characterized. In agreement with the pattern of secondary structure changes detected by circular dichroism described in the accompanying paper (DOI 10.1021/bi I 00338e), tertiary structure changes are distinct over four SDS concentration ranges based on the expected predominant micellar structures. Dodecyl maltoside (DM)/SDS mixed micelle spheres (0.05-0.3% SDS) turn into SDS spheres (0.3-3% SDS) that gradually (3-15% SDS) become cylindrical (above 15% SDS). Denatured states in SDS spheres and cylinders show a relatively greater burial of cysteine and tryptophan residues and are more compact as compared to the states observed in mixed micellar structures. Protein structural changes at the membrane/water interface region are most prominent at very low SDS concentrations but reach transient stability in the compact conformations in SDS spheres. This is the first experimental evidence for the formation of a compact unfolding intermediate state with flexible surface elements in a mem-brane protein.
机译：关于膜蛋白未折叠状态的残留结构的分子性质知之甚少。随附论文（DOI 0.102 I / bil00338e）中所述的化学变性剂筛选方法，可最大限度地展开哺乳动物膜蛋白和原型G蛋白偶联受体视紫红质，而不会受到聚集的干扰，可鉴定出十二烷基硫酸钠（SDS）单独或组合使用与其他化学品一起使用，作为最合适的变性剂。在这里，我们启动视紫红质的SDS变性状态的生物物理表征。使用吸收，稳态和时间分辨荧光光谱，动态光散射和半胱氨酸可及性研究，表征了变性态的三级结构。与随附文件（DOI 10.1021 / bi I 00338e）中所述的通过圆二色性检测到的二级结构变化的模式一致，基于预期的主要胶束结构，三级结构变化在四个SDS浓度范围内是不同的。十二烷基麦芽糖苷（DM）/ SDS混合胶束球（0.05-0.3％SDS）转变为SDS球（0.3-3％SDS），然后逐渐（3-15％SDS）变成圆柱形（高于15％SDS）。与在混合胶束结构中观察到的状态相比，SDS球体和圆柱体中的变性状态显示出相对较高的半胱氨酸和色氨酸残基埋葬，并且更紧凑。在非常低的SDS浓度下，膜/水界面区域的蛋白质结构变化最为明显，但在SDS球体的紧凑构象中达到了短暂的稳定性。这是在膜蛋白中形成具有柔性表面元件的致密展开中间状态的第一个实验证据。

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