Gabellieri E, Strambini G B, Baracca A, Solaini G
Consiglio Nazionale delle Richerche, Istituto di Biofisica, Pisa, Italy.
Biophys J. 1997 Apr;72(4):1818-27. doi: 10.1016/S0006-3495(97)78828-3.
Phosphorescence and fluorescence energy transfer measurements have been used to locate the epsilon-subunit within the know structural frame of the mitochondrial soluble part of F-type H(+)-ATPase complex (F1). The fluorescence probe 2'-O-(trinitrophenyl)adenosine-5'-triphosphate was bound to the nucleotide binding sites of the enzyme, whereas the probe 7-diethylamino-3'-(4'-maleimidylphenyl)-4-methylcoumarin was attached to the single sulfhydryl residue of isolated oligomycin sensitivity-conferring protein (OSCP), which was then reconstituted with F1. Fluorescence and phosphorescence resonance energy transfer yields from the lone tryptophan residue of F1 present in the epsilon-polypeptide and the fluorescence labels attached to the F1 complex established that tryptophan is separated by 3.7 nm from Cys-118 of OSCP in the reconstituted OSCP-F1 complex, by 4.9 nm from its closest catalytic site and by more than 6.4 nm from the two other catalytic sites, including the lowest affinity ATP site. These separations together with the crystallographic coordinates of the F1 complex (Abrahams, J.P., A. G. W. Leslie, R. Lutter, and J.E. Walker. 1994. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature. 370:621-628) place the epsilon-subunit in the stem region of the F1 molecule in a unique asymmetrical position relative to the catalytic sites of the enzyme.
磷光和荧光能量转移测量已被用于在线粒体F型H(+)-ATP酶复合体（F1）可溶性部分的已知结构框架内定位ε亚基。荧光探针2'-O-(三硝基苯基)腺苷-5'-三磷酸与该酶的核苷酸结合位点结合，而探针7-二乙氨基-3'-(4'-马来酰亚胺基苯基)-4-甲基香豆素则连接到分离的赋予寡霉素敏感性的蛋白质（OSCP）的单个巯基残基上，然后将其与F1重组。来自存在于ε多肽中的F1的唯一色氨酸残基以及连接到F1复合体上的荧光标记的荧光和磷光共振能量转移产率表明，在重组的OSCP-F1复合体中，色氨酸与OSCP的Cys-118相距3.7 nm，与最接近的催化位点相距4.9 nm，与包括最低亲和力ATP位点在内的其他两个催化位点相距超过6.4 nm。这些距离连同F1复合体的晶体学坐标（Abrahams, J.P., A. G. W. Leslie, R. Lutter, and J.E. Walker. 1994. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature. 370:621-628）将ε亚基置于F1分子的茎区，相对于该酶的催化位点处于独特的不对称位置。
