Castello P R, González Flecha F L, Caride A J, Fernández H N, Delfino J M, Rossi J P
Departamento de Química Biológica-IQUIFIB, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
Protein Sci. 1997 Aug;6(8):1708-17. doi: 10.1002/pro.5560060811.
A systematic study of the membrane-associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine ([125I]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane-bound probe, [3H]bis-phosphatidylethanolamine (trifluoromethyl) phenyldiazirine. A significant light-dependent labeling of an M(r) 135,000-140,000 peptide, corresponding to the full Ca2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments by SDS-PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with [125I]TID, either in isolated red blood cell membranes or after the enzyme was purified, yields a similar pattern of labeled peptides. Taken together, these results validate the use of either probe to study the lipid interface of the membrane-embedded region of this protein, and sustain the notion that the conformation of the pump is maintained throughout the procedures of solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis on a detailed analysis of the N-terminal domain of the Ca2+ pump. A labeled peptide of M(r) 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [125I]TID to proteolytic fragments pertaining to the amino-terminal region indicates the existence of two transmembrane stretches in this domain. A theoretical analysis based on the amino acid sequence 1-322 predicts two segments with high probability of membrane insertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity and variability compatible with alpha-helical structure. These results strongly suggest the existence of a transmembrane helical hairpin motif near the N-terminus of the Ca2+ pump.
通过疏水光标记法对红细胞质膜钙泵中与膜相关的区域进行了系统研究。纯化的钙泵用3 -（三氟甲基）-3 -（间-[¹²⁵I]碘苯基）-重氮甲烷（[¹²⁵I]TID）进行标记，这是一种通用的可光活化疏水探针。将这些结果与用严格膜结合探针[³H]双磷脂酰乙醇胺（三氟甲基）苯基重氮甲烷标记的酶进行比较。两种探针均观察到与完整钙泵相对应的M（r）135,000 - 140,000肽段有明显的光依赖性标记。在用每种探针标记泵并通过SDS - PAGE分离片段后，观察到标记肽段的共同模式。同样，无论是在分离的红细胞膜中还是在酶纯化后，用[¹²⁵I]TID标记钙泵都产生相似的标记肽段模式。综上所述，这些结果证实了使用任何一种探针来研究该蛋白质膜嵌入区域的脂质界面的可行性，并支持了在溶解、亲和纯化和重组到蛋白脂质体的整个过程中泵的构象得以维持的观点。在这项工作中，我们特别强调对钙泵N端结构域的详细分析。纯化了属于该区域的M（r）40,000标记肽段，并用V8蛋白酶进一步消化。[¹²⁵I]TID特异性掺入到与氨基末端区域相关的蛋白水解片段中，表明该结构域存在两个跨膜区段。基于氨基酸序列1 - 322的理论分析预测有两个具有高膜插入概率的区段，与实验数据一致。每个区段都显示出与α - 螺旋结构兼容的疏水性和变异性的周期性模式。这些结果强烈表明在钙泵N端附近存在跨膜螺旋发夹基序。
