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Zinc binding of Tim10: Evidence for existence of an unstructured binding intermediate for a zinc finger protein
Author(s) -
Ivanova Ekaterina,
Ball Matthew,
Lu Hui
Publication year - 2008
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21713
Subject(s) - zinc , chemistry , zinc finger , fluorescence , molten globule , circular dichroism , crystallography , biophysics , binding site , biochemistry , biology , organic chemistry , physics , quantum mechanics , transcription factor , gene
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 × 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 (∼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. Proteins 2008. © 2007 Wiley‐Liss, Inc.