Baracca A, Gabellieri E, Barogi S, Solaini G
Dipartimento di Biochimica G. Moruzzi, Università di Bologna, Italy.
J Biol Chem. 1995 Sep 15;270(37):21845-51. doi: 10.1074/jbc.270.37.21845.
Changes in conformation of the epsilon-subunit of the bovine heart mitochondrial F1-ATPase complex as a result of nucleotide binding have been demonstrated from the phosphorescence emission of tryptophan. The triplet state lifetime shows that whereas nucleoside triphosphate binding to the enzyme in the presence of Mg2+ increases the flexibility of the protein structure surrounding the chromophore, nucleoside diphosphate acts in an opposite manner, enhancing the rigidity of this region of the macromolecule. Such changes in dynamic structure of the epsilon-subunit are evident at high ligand concentration added to both the nucleotide-depleted F1 (Nd-F1) and the F1 preparation containing the three tightly bound nucleotides (F1(2,1)). Since the effects observed are similar in both the F1 forms, the binding to the low affinity sites must be responsible for the conformational changes induced in the epsilon-subunit. This is partially supported by the observation that the Trp lifetime is not significantly affected by adding an equimolar concentration of adenine nucleotide to Nd-F1. The effects on protein structure of nucleotide binding to either catalytic or noncatalytic sites have been distinguished by studying the phosphorescence emission of the F1 complex prepared with the three noncatalytic sites filled and the three catalytic sites vacant (F1(3,0)). Phosphorescence lifetime measurements on this F1 form demonstrate that the binding of Mg-NTP to catalytic sites induces a slight enhancement of the rigidity of the epsilon-subunit. This implies that the binding to the vacant noncatalytic site of F1(2,1) must exert the opposite and larger effect of enhancing the flexibility of the protein structure observed in both Nd-F1 and F1(2,1). The observation that enhanced flexibility of the protein occurs upon addition of adenine nucleotides to F1(2,1) in the absence of Mg2+ provides direct support for this suggestion. The connection between changes in structure and the possible functional role of the epsilon-subunit is discussed.
通过色氨酸的磷光发射已证明，由于核苷酸结合，牛心线粒体F1 - ATP酶复合体的ε亚基构象发生了变化。三重态寿命表明，在Mg2 +存在下，核苷三磷酸与酶的结合增加了发色团周围蛋白质结构的灵活性，而核苷二磷酸则以相反的方式起作用，增强了大分子该区域的刚性。在添加到核苷酸耗尽的F1（Nd - F1）和含有三个紧密结合核苷酸的F1制剂（F1(2,1)）中的高浓度配体情况下，ε亚基动态结构的这种变化很明显。由于在两种F1形式中观察到的效应相似，与低亲和力位点的结合必定是ε亚基中诱导构象变化的原因。向Nd - F1中添加等摩尔浓度的腺嘌呤核苷酸时，色氨酸寿命未受到显著影响，这一观察结果部分支持了上述观点。通过研究用三个非催化位点填充且三个催化位点空缺的F1复合体（F1(3,0)）的磷光发射，区分了核苷酸与催化或非催化位点结合对蛋白质结构的影响。对这种F1形式的磷光寿命测量表明，Mg - NTP与催化位点的结合会导致ε亚基刚性略有增强。这意味着与F1(2,1)的空缺非催化位点的结合必定产生相反且更大的效应，即增强在Nd - F1和F1(2,1)中观察到的蛋白质结构的灵活性。在不存在Mg2 +的情况下，向F1(2,1)中添加腺嘌呤核苷酸后蛋白质灵活性增强的观察结果为这一推测提供了直接支持。文中讨论了结构变化与ε亚基可能的功能作用之间的联系。
