PMID:10736187
| Citation |
Kersten, MV, Dunn, SD, Wise, JG and Vogel, PD (2000) Site-directed spin-labeling of the catalytic sites yields insight into structural changes within the F0F1-ATP synthase of Escherichia coli. Biochemistry 39:3856-60 |
|---|---|
| Abstract |
Electron spin resonance (ESR) spectroscopy using site-specific cysteine spin-labeling of the catalytic nucleotide binding sites of F(1)-ATPase was employed to investigate conformational changes within the nucleotide binding sites of the enzyme. Mutant Escherichia coli F(1) that had been modified at position beta-Y331C with a spin label showed almost normal catalytic activity and enabled us to study the effects of binding of different nucleotides and of the F(o) subunit b on the conformation of the catalytic binding sites. The ESR spectra of the spin-labeled, nucleotide-depleted F(1) indicate asymmetry within the sites as is expected from the structural models of the enzyme. Nucleotide binding to the enzyme clearly affects the conformation of the sites; the most pronounced feature upon nucleotide binding is the formation of catalytic site(s) in a very open conformation. Using the same beta-331 spin-labeled F(1) and a truncated form of F(o) subunit b, b(24)(-)(156), we found that binding of b(24)(-)(156) to spin-labeled F(1) significantly changes the conformation of the catalytic sites. In this paper we present data that for the first time directly show that a conformational binding change takes place upon binding of nucleotides to the nucleotide binding sites and that also show that binding of b(24)(-)(156) strongly affects the conformation of the catalytic sites, most likely by increasing the population of binding sites that are in the open conformation. |
| Links | |
| Keywords |
Adenosine Diphosphate/chemistry; Adenosine Triphosphate/chemistry; Adenylyl Imidodiphosphate/chemistry; Binding Sites/genetics; Catalysis; Cyclic N-Oxides/chemistry; Cysteine/chemistry; Cysteine/genetics; Electron Spin Resonance Spectroscopy; Escherichia coli/enzymology; Escherichia coli/genetics; Imidazoles/chemistry; Imidazolidines; Magnesium/chemistry; Mutagenesis, Site-Directed; Protein Conformation; Proton-Translocating ATPases/chemistry; Proton-Translocating ATPases/genetics; Proton-Translocating ATPases/metabolism; Spin Labels |
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