Binding of the b-subunit in the ATP synthase from Escherichia coli.
Binding of the b-subunit in the ATP synthase from Escherichia coli.
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大肠杆菌 ATP 合酶中 b 亚基的结合。
DOI：
10.1021/bi0357098
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发表时间：
2004
期刊：
影响因子：
2.9
通讯作者：
P. Gräber
中科院分区：
文献类型：
--
作者：
M. Diez;M. Börsch;B. Zimmermann;P. Turina;S. Dunn;P. Gräber
ATP 合酶的旋转机制需要定子亚基内的牢固结合。在这项工作中，我们研究了 b 亚基与大肠杆菌 F(1)-ATP 酶的结合亲和力。通过分析超速离心研究了不含氨基酸 1-33 的截短 b 亚基 b(34-156)T62C 的二聚化，结果解离常数为 1.8 µM。通过荧光相关光谱和稳态荧光研究了 b 亚基单体和二聚体形式与分离的 F(1) 部分的结合。突变体 b(34-156)T62C 和 EF(1)-gammaT106C 用多种荧光团标记。使用荧光相关光谱来测量标记的b-亚基、标记的F(1)以及标记的b-亚基与未标记的F(1)的混合物的平移扩散时间。数据分析显示F(1)b(2)复合物的解离常数为0.2 nM，产生的吉布斯结合自由能为DeltaG(o)= -55 kJ mol(-1)。在稳态荧光共振能量转移 (FRET) 测量中，发现 b 亚基与 EF(1)-gammaT106C-Alexa488 的结合导致荧光强度降低了初始 FRET 供体荧光强度的三分之一。荧光的减少被测量为b浓度的函数，并且数据通过包括b亚基二聚化和b和b(2)与F(1)结合的平衡的模型来描述。为了定量描述荧光减少，我们使用了两种不同的模型：第一个和第二个 b 亚基的结合导致相同的荧光减少（模型 1），或者仅第一个 b 亚基的结合导致荧光减少（模型 2）。数据评估显示 F(1)b(2) 复合物的解离常数为 0.6 nM（模型 1）或 14 nM（模型 2），分别给出 DeltaG(o)= -52 kJ mol(-1) 和 DeltaG(o)= -45 kJ mol(-1)。细胞中 ATP 合成观察到的最大 DeltaG 约为 DeltaG= 55 kJ mol(-1)。因此，对于 ATP 合成的自由能在转子和定子亚基之间的弹性应变中积累，然后一步转换到催化位点的模型来说，b 亚基的结合能似乎太低。能量转换至少发生两个步骤的模型受到青睐。
The rotary mechanism of ATP synthase requires a strong binding within stator subunits. In this work we studied the binding affinity of the b-subunit to F(1)-ATPase of Escherichia coli. The dimerization of the truncated b-subunit without amino acids 1-33, b(34-156)T62C, was investigated by analytical ultracentrifugation, resulting in a dissociation constant of 1.8 microM. The binding of b-subunit monomeric and dimeric forms to the isolated F(1) part was investigated by fluorescence correlation spectroscopy and steady-state fluorescence. The mutants b(34-156)T62C and EF(1)-gammaT106C were labeled with several fluorophores. Fluorescence correlation spectroscopy was used to measure translational diffusion times of the labeled b-subunit, labeled F(1), and a mixture of the labeled b-subunit with unlabeled F(1). Data analysis revealed a dissociation constant of 0.2 nM of the F(1)b(2) complex, yielding a Gibbs free energy of binding of DeltaG(o)= -55 kJ mol(-1). In steady-state fluorescence resonance energy transfer (FRET) measurements it was found that binding of the b-subunit to EF(1)-gammaT106C-Alexa488 resulted in a fluorescence decrease of one-third of the initial FRET donor fluorescence intensity. The decrease of fluorescence was measured as a function of b-concentration, and data were described by a model including equilibria for dimerization of the b-subunit and binding of b and b(2) to F(1). For a quantitative description of fluorescence decrease we used two different models: the binding of the first and the second b-subunit causes the same fluorescence decrease (model 1) or only the binding of the first b-subunit causes fluorescence decrease (model 2). Data evaluation revealed a dissociation constant for the F(1)b(2) complex of 0.6 nM (model 1) or 14 nM (model 2), giving DeltaG(o)= -52 kJ mol(-1) and DeltaG(o)= -45 kJ mol(-1), respectively. The maximal DeltaG observed for ATP synthesis in cells is approximately DeltaG= 55 kJ mol(-1). Therefore, the binding energy of the b-subunit seems to be too low for models in which the free energy for ATP synthesis is accumulated in the elastic strain between rotor and stator subunits and then transduced to the catalytic site in one single step. Models in which energy transduction takes place in at least two steps are favored.