Richter M L, Snyder B, McCarty R E, Hammes G G
Biochemistry. 1985 Oct 8;24(21):5755-63. doi: 10.1021/bi00342a011.
Fluorescent probes were attached to the single sulfhydryl residue on the isolated epsilon polypeptide of chloroplast coupling factor 1 (CF1), and the modified polypeptide was reconstituted with the epsilon-deficient enzyme. A binding stoichiometry of one epsilon polypeptide per CF1 was obtained. This stoichiometry corresponded to a maximum inhibition of the Ca2+-dependent ATPase activity of the enzyme induced by epsilon removal. Resonance energy transfer between the modified epsilon polypeptide and fluorescent probes attached to various other sites on the enzyme allowed distance measurements between these sites and the epsilon polypeptide. The epsilon-sulfhydryl is nearly equidistant from both the disulfide (23 A) and the dark-accessible sulfhydryl (26 A) of the gamma subunit. Measurement of the distance between epsilon and the light-accessible gamma-sulfhydryl was not possible due to an apparent exclusion of modified epsilon from epsilon-deficient enzyme after modification of the light-accessible site. The distances measured between epsilon and the nucleotide binding sites on the enzyme were 62, 66, and 49 A for sites 1, 2, and 3, respectively. These measurements place the epsilon subunit in close physical proximity to the sulfhydryl-containing domains of the gamma subunit and approximately 40 A from the membrane surface. Enzyme activity measurements also indicated a close association between the epsilon and gamma subunits: epsilon removal caused a marked increase in accessibility of the gamma-disulfide bond to thiol reagents and exposed a trypsin-sensitive site on the gamma subunit. Either disulfide bond reduction or trypsin cleavage of gamma significantly enhanced the Ca2+-ATPase activity of the epsilon-deficient enzyme. Thus, the epsilon and gamma polypeptides of coupling factor 1 are closely linked, both physically and functionally.
荧光探针连接到叶绿体偶联因子1（CF1）分离出的ε多肽上的单个巯基残基上，然后将修饰后的多肽与缺乏ε的酶进行重组。得到每个CF1含有一个ε多肽的结合化学计量比。该化学计量比对应于去除ε后酶的Ca2+依赖性ATP酶活性的最大抑制。修饰后的ε多肽与连接在酶上其他不同位点的荧光探针之间的共振能量转移，使得能够测量这些位点与ε多肽之间的距离。ε-巯基与γ亚基的二硫键（23 Å）和暗可及巯基（26 Å）的距离几乎相等。由于在修饰光可及位点后，修饰后的ε明显被缺乏ε的酶排斥，因此无法测量ε与光可及γ-巯基之间的距离。酶上ε与核苷酸结合位点1、2和3之间测量的距离分别为62 Å、66 Å和49 Å。这些测量结果表明，ε亚基在物理位置上与γ亚基含巯基的结构域紧密相邻，且距离膜表面约40 Å。酶活性测量还表明ε和γ亚基之间存在紧密联系：去除ε会导致γ-二硫键对硫醇试剂的可及性显著增加，并暴露出γ亚基上一个对胰蛋白酶敏感的位点。γ的二硫键还原或胰蛋白酶切割都会显著增强缺乏ε的酶的Ca2+-ATP酶活性。因此，偶联因子1的ε和γ多肽在物理和功能上都紧密相连。
