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首页> 外文期刊>Biochemistry >Topological disposition of Cys 222 in the alpha-subunit of nicotinic acetylcholine receptor analyzed by fluorescence-quenching and electron paramagnetic resonance measurements
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Topological disposition of Cys 222 in the alpha-subunit of nicotinic acetylcholine receptor analyzed by fluorescence-quenching and electron paramagnetic resonance measurements

机译：通过荧光猝灭和电子顺磁共振测量分析烟碱乙酰胆碱受体α-亚基中Cys 222的拓扑结构

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摘要

The structure of the nicotinic acetylcholine receptor (AChR) has been studied using a combination of fluorescence quenching and electron paramagnetic resonance (EPR) collision gradient methods. The AChR from Torpedo californica was labeled with a fluorescent probe, N-(1-pyrenyl)-maleimide, specific for sulfhydryls in a hydrophobic environment, under conditions of selective labeling of Cys222 in the alpha-subunit. alpha Cys222 is located in the postulated M1 transmembrane domain and predicted to be at the center of an alpha-helical secondary structure, The spatial disposition of the acetylcholine receptor-bound pyrene with respect to the membrane bilayer was assessed by fluorescence quenching measurements. Quenching of pyrene fluorescence by spin-labeled fatty acids with the doxyl group at positions C-5 and C-12 revealed that the former was more effective, suggesting that the fluorophore is located closer to the membrane-water interface than to the hydrophobic interior. Power saturation EPR spectroscopy was also used to examine the effect of molecular oxygen and water-soluble paramagnetic reagents on the saturation behavior of a nitroxide spin label, which was specifically attached to the same alpha Cys222 residue. Using the gradients of these paramagnetic reagents through the membrane-solution interface, the distance for the nitroxide derivative from the membrane-solution interface was measured to be approximately 7 Angstrom from the headgroup region of the phospholipid bilayer, in agreement with fluorescence quenching results. These results suggest that the M1 transmembrane domain of the AChR probably forms an irregular structure, a beta-strand, or an alpha-helical structure that may span the membrane in a way different from a linear alpha-helix. [References: 52]
机译：烟酸乙酰胆碱受体（AChR）的结构已使用荧光猝灭和电子顺磁共振（EPR）碰撞梯度方法相结合进行了研究。在选择性标记α-亚基中Cys222的条件下，用疏水探针特异于巯基的荧光探针N-（1-吡啶基）-马来酰亚胺标记来自加州鱼雷的AChR。 αCys222位于假定的M1跨膜结构域中，并预计位于α螺旋二级结构的中心。通过荧光猝灭测量评估乙酰胆碱受体结合的pyr相对于膜双层的空间布置。旋转标记的脂肪酸在位置C-5和C-12处带有二甲苯基基团对pyr荧光进行猝灭，表明前者更有效，这表明荧光团更靠近膜-水界面，而不是疏水内部。功率饱和EPR光谱仪还用于检查分子氧和水溶性顺磁性试剂对一氧化氮自旋标记物的饱和行为的影响，该标记特异地附着在相同的alpha Cys222残基上。使用这些顺磁性试剂通过膜-溶液界面的梯度，从膜-溶液界面到氮氧化物衍生物的距离被测量为距磷脂双层的头基区域约7埃，这与荧光猝灭结果一致。这些结果表明，AChR的M1跨膜结构域可能形成不规则结构，β链或α螺旋结构，可能以不同于线性α螺旋的方式跨越膜。 [参考：52]

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