Shahrokh Z, Verkman A S, Shohet S B
Department of Laboratory Medicine, University of California, San Francisco 94143.
J Biol Chem. 1991 Jun 25;266(18):12082-9.
To assess the molecular architecture of the human erythrocyte skeletal protein 4.1:bilayer interface, the distance between a donor sulfhydryl-specific fluorescent probe attached to a region near the glycophorin-binding domain of protein 4.1 and an acceptor lipophilic probe in the exposed leaflet of inside-out vesicles (IOVs) was measured by fluorescence resonance energy transfer. To prevent aggregation and loss of function, protein 4.1 was labeled in situ on the surface of IOVs, purified, and rebound onto fresh IOVs. The labeled protein 4.1 was similar to the native protein in its gel electrophoretic pattern and its binding affinity to stripped-IOVs (Kd 35 +/- 4 nM). Energy transfer was assessed using two donor-acceptor pairs, 5-[2-[(iodoacetyl)amino]ethyl] aminonaphthalene-1-sulfonic acid and 3,3'-ditetradecyloxacarbocyanine perchlorate, or 5-iodoacetamidofluorescein and tetramethylrhodamine phosphatidylethanolamine. Using both donor fluorescence intensity and lifetime quenching measurements, an average distance of 75 +/- 5 A between the probe on the protein and the surface of IOVs was found. In parallel fluorescence resonance energy transfer studies with protein 4.1 and liposomes with a phospholipid composition similar to the inner leaflet of the red cell membrane, a closer distance was found (49 +/- 5 A). Two control experiments validated energy transfer: (a) the spectrum of a mixture of IOVs separately labeled with donor and acceptor was different from the spectrum of the doubly labeled IOVs at identical donor and acceptor concentrations; and (b) no energy transfer was observed following detergent disruption of the geometric relationship between donor and acceptor. Taken together, these observations suggest that membrane-bound protein 4.1 is elongated and that the labeled site is located at a position deep in the 30-kDa N-terminal glycophorin-binding domain of the protein. The data are also consistent with the view that the cytoplasmic tail of glycophorin is interposed between protein 4.1 and the lipids. These experiments represent the first measurement of a distance between a skeletal protein and the lipid bilayer.
为评估人红细胞骨架蛋白4.1与双层膜界面的分子结构，通过荧光共振能量转移测量了连接到蛋白4.1糖蛋白结合结构域附近区域的供体巯基特异性荧光探针与内翻囊泡（IOV）外表面叶中的受体亲脂性探针之间的距离。为防止聚集和功能丧失，蛋白4.1在IOV表面原位标记、纯化，然后重新结合到新鲜的IOV上。标记后的蛋白4.1在凝胶电泳图谱及其与脱蛋白IOV的结合亲和力（Kd 35±4 nM）方面与天然蛋白相似。使用两个供体-受体对评估能量转移，即5-[2-[(碘乙酰基)氨基]乙基]氨基萘-1-磺酸和3,3'-二四癸基氧杂羰花青高氯酸盐，或5-碘乙酰胺荧光素和四甲基罗丹明磷脂酰乙醇胺。通过供体荧光强度和寿命猝灭测量，发现蛋白上的探针与IOV表面之间的平均距离为75±5 Å。在与蛋白4.1和具有类似于红细胞膜内叶磷脂组成的脂质体进行的平行荧光共振能量转移研究中，发现了更近的距离（49±5 Å）。两项对照实验验证了能量转移：（a）在相同供体和受体浓度下，分别用供体和受体标记的IOV混合物的光谱与双重标记的IOV的光谱不同；（b）去污剂破坏供体和受体之间的几何关系后未观察到能量转移。综上所述，这些观察结果表明膜结合的蛋白4.1是伸长的，并且标记位点位于该蛋白30 kDa N端糖蛋白结合结构域深处的位置。数据也与糖蛋白的细胞质尾巴插入蛋白4.1和脂质之间的观点一致。这些实验代表了对骨架蛋白与脂质双层之间距离的首次测量。
