title: Zinc binding of Tim10: evidence for existence of an unstructured binding intermediate for a zinc finger protein.
abstract: Zinc-finger proteins are among the most abundant proteins in eukaryotic genomes. Tim10 and all the small Tim proteins of the mitochondrial intermembrane space contain a consensus twin CX(3)C zinc-finger motif. Zn(2+) can bind to the reduced Tim10, but not disulphide bonded (oxidized) protein. However, the zinc-binding reaction of Tim10 and of zinc-finger proteins, in general, is ill-defined. In this study, the thermodynamic and kinetic properties of zinc-binding to reduced Tim10 were investigated using circular dichroism (CD), fluorescence spectrometry, and stopped-flow fluorescence techniques. At equilibrium, coupled with the use of protein fluorescence and metal chelators, the zinc-binding affinity was determined for Tim10 to be about 8 x 10(-10)M. Then, far UV CD was used to investigate the secondary structure change upon zinc-binding of the same set of protein samples at various free Zn(2+) concentrations. Comparison between the results of CD and fluorescence studies showed that the zinc-binding reaction is not a simple one-step process. It involves formation of a binding intermediate that is structurally as unfolded as the apoTim10; subsequently, a degree of folding is induced at increased zinc concentrations in the final complex. Next, the stopped-flow fluorescence technique was used to investigate the kinetic process of the binding reaction. Data analysis shows that the reaction has a single kinetic phase at a low free Zn(2+) concentration ( approximately 1 nM), and a double kinetic phase at a high free Zn(2+) concentration. The kinetic result is consistent with that of the studies at equilibrium. Therefore, a two-step reaction model mechanism is proposed, in which zinc-binding is regulated by the initial selective-binding of Zn(2+) to Cys followed by folding. Implication of the two-step zinc-binding mechanism for Zn(2+) trafficking in the cell is discussed.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Manchester M13 9PT, United Kingdom.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Ivanova
authors.forename: Matthew
authors.initials: M
authors.lastname: Ball
authors.forename: Hui
authors.initials: H
authors.lastname: Lu
mesh_terms: D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D024101:Mitochondrial Proteins / Q000737:chemistry; D018832:Molecular Chaperones / Q000737:chemistry*; D011485:Protein Binding; D017510:Protein Folding; D013057:Spectrum Analysis; D015032:Zinc / Q000737:chemistry*; D016335:Zinc Fingers
chemical_list: D029968:Escherichia coli Proteins; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; C478387:TIM10 protein, E coli; D015032:Zinc