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首页> 外文期刊>Chemistry: A European journal >Regioselective Photolabeling of Glycophorin A in Membranes
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Regioselective Photolabeling of Glycophorin A in Membranes

机译：膜中糖蛋白A的区域选择性光标记

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摘要

We have developed a chemical method for directly identifying the amino acid residues of the transmembrane domain of a protein that are located right in the center of the membrane. Glycophorin A (GPA), the major sialoglycoprotein of human erythrocytes, was the first membrane protein whose primary sequence was elucidated, but its three-dimensional structure is still not known. GPA has been reconstituted into liposomes formed from dimyristopylphosphatidylcholine, dimyristoylphopshatidylserine, cholesterol, and a bola-amphiphilic phospholipidic photoactivatable probe (radioactive probe 1) by a detergent-mediated method. Electron microscopy confirmed the formation of spherical vesicular structures, and sucrose-density gradients revealed that the proteoliposomes comprised only one membrane fraction. Proteinase-K digestion of GPA in the proteoliposomes suggested that the orientation of GPA in reconstituted proteoliposomes was virtually identical to that observed in natural erythrocyte membranes. After photo-irradiation of the reconstituted proteoliposomes and in situ tryptic digestioh, the photolableed amino acid residuces were analyzed by Edman degradation and their radioactivity was measured. Va180 and Met81, which had been assumed to be located near the center of the transmembrane domain of GPA, were indeed highly selectively photolabeled by probe 1. The new method might be appleid to analyze the three-dimensional arrangement of the transmembrane domain of protein complexes that are made up from several subunits.
机译：我们已经开发出一种化学方法，可以直接识别位于膜中心的蛋白质跨膜结构域的氨基酸残基。糖蛋白A（GPA）是人类红细胞的主要唾液糖蛋白，是第一个阐明其一级序列的膜蛋白，但其三维结构仍不清楚。 GPA已通过去污剂介导的方法重组为由二肉豆蔻酰磷脂酰胆碱，二肉豆蔻酰磷脂酰丝氨酸，胆固醇和硼酸两亲性磷脂可光活化探针（放射性探针1）形成的脂质体。电子显微镜检查证实了球形水泡结构的形成，蔗糖密度梯度表明该蛋白脂质体仅包含一个膜部分。蛋白脂质体中GPA的蛋白酶K消化表明，重组蛋白脂质体中GPA的方向实际上与天然红细胞膜中观察到的相同。在对重组蛋白脂质体进行光辐照和原位胰蛋白酶消化后，通过Edman降解分析可光化的氨基酸残基，并测量其放射性。 Va180和Met81被假定位于GPA跨膜结构域的中心附近，实际上已被探针1高度选择性地光标记。这种新方法可能是小应用程序，可以分析蛋白质复合物跨膜结构域的三维排列由几个子单元组成。

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