Binding of the b-Subunit in the ATP Synthase from Escherichia coli
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- 作者：Manuel Diez ; Michael Bö ; rsch ; Boris Zimmermann ; Paola Turina ; Stanley D. Dunn ; and Peter Grä ; ber
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：February 3, 2004
- 年：2004
- 卷：43
- 期：4
- 页码：1054 - 1064
- 全文大小：183K
- 年卷期：v.43,no.4(February 3, 2004)
- ISSN：1520-4995
文摘
The rotary mechanism of ATP synthase requires a strong binding within stator subunits. Inthis work we studied the binding affinity of the b-subunit to F1-ATPase of Escherichia coli. The dimerizationof the truncated b-subunit without amino acids 1-33, b34-156T62C, was investigated by analyticalultracentrifugation, resulting in a dissociation constant of 1.8 
M. The binding of b-subunit monomericand dimeric forms to the isolated F1 part was investigated by fluorescence correlation spectroscopy andsteady-state fluorescence. The mutants b34-156T62C and EF1-
T106C were labeled with severalfluorophores. Fluorescence correlation spectroscopy was used to measure translational diffusion times ofthe labeled b-subunit, labeled F1, and a mixture of the labeled b-subunit with unlabeled F1. Data analysisrevealed a dissociation constant of 0.2 nM of the F1b2 complex, yielding a Gibbs free energy of bindingof 
G
= -55 kJ mol-1. In steady-state fluorescence resonance energy transfer (FRET) measurementsit was found that binding of the b-subunit to EF1-T106C-Alexa488 resulted in a fluorescence decreaseof one-third of the initial FRET donor fluorescence intensity. The decrease of fluorescence was measuredas a function of b-concentration, and data were described by a model including equilibria for dimerizationof the b-subunit and binding of b and b2 to F1. For a quantitative description of fluorescence decrease weused two different models: the binding of the first and the second b-subunit causes the same fluorescencedecrease (model 1) or only the binding of the first b-subunit causes fluorescence decrease (model 2).Data evaluation revealed a dissociation constant for the F1b2 complex of 0.6 nM (model 1) or 14 nM(model 2), giving G = -52 kJ mol-1 and G = -45 kJ mol-1, respectively. The maximal G observedfor ATP synthesis in cells is approximately G = 55 kJ mol-1. Therefore, the binding energy of theb-subunit seems to be too low for models in which the free energy for ATP synthesis is accumulated inthe elastic strain between rotor and stator subunits and then transduced to the catalytic site in one singlestep. Models in which energy transduction takes place in at least two steps are favored.
M. The binding of b-subunit monomericand dimeric forms to the isolated F1 part was investigated by fluorescence correlation spectroscopy andsteady-state fluorescence. The mutants b34-156T62C and EF1-T106C were labeled with severalfluorophores. Fluorescence correlation spectroscopy was used to measure translational diffusion times ofthe labeled b-subunit, labeled F1, and a mixture of the labeled b-subunit with unlabeled F1. Data analysisrevealed a dissociation constant of 0.2 nM of the F1b2 complex, yielding a Gibbs free energy of bindingof G = -55 kJ mol-1. In steady-state fluorescence resonance energy transfer (FRET) measurementsit was found that binding of the b-subunit to EF1-T106C-Alexa488 resulted in a fluorescence decreaseof one-third of the initial FRET donor fluorescence intensity. The decrease of fluorescence was measuredas a function of b-concentration, and data were described by a model including equilibria for dimerizationof the b-subunit and binding of b and b2 to F1. For a quantitative description of fluorescence decrease weused two different models: the binding of the first and the second b-subunit causes the same fluorescencedecrease (model 1) or only the binding of the first b-subunit causes fluorescence decrease (model 2).Data evaluation revealed a dissociation constant for the F1b2 complex of 0.6 nM (model 1) or 14 nM(model 2), giving G = -52 kJ mol-1 and G = -45 kJ mol-1, respectively. The maximal G observedfor ATP synthesis in cells is approximately G = 55 kJ mol-1. Therefore, the binding energy of theb-subunit seems to be too low for models in which the free energy for ATP synthesis is accumulated inthe elastic strain between rotor and stator subunits and then transduced to the catalytic site in one singlestep. Models in which energy transduction takes place in at least two steps are favored.