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Biochemistry. 2009 Apr 7;48(13):2950-9. doi: 10.1021/bi802072u.

Metal binding kinetics of bi-histidine sites used in psi analysis: evidence of high-energy protein folding intermediates.

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The Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.


The zinc-specific fluorophore, Zinpyr-1, is used in competition assays to determine the kinetic and thermodynamic parameters of Zn2+ binding to engineered bi-histidine sites located in ubiquitin and the B domain of protein A (BdpA). These binding sites are used in psi analysis studies to investigate structure formation in the folding transition state identified by the change in folding rate upon addition of metal ions. For ubiquitin, the on-rate binding constant and binding affinity for a site located along an alpha-helix are measured to be approximately 10(7) M-1 s-1 and 3 microM, respectively. For a site located across two beta-strands, the metal binding affinity was too weak to measure in the dye competition assays (Kd > 55 microM). The equilibrium-determined values for the Zn2+-induced stabilization of ubiquitin and BdpA match the values derived from changes in the global folding and unfolding rates. Therefore, metal ion binding is in fast equilibrium during the transit over the free energy barrier. Accordingly, the folding rate must be slower than the product of the fractional population of a high-energy intermediate with the metal site formed and the metal binding on-rate constant. The known folding rate of 20 s-1 at 1.5 M guanidinium chloride in 400 microM Zn2+ provides an upper bound for the stability of such intermediates (DeltaG(U-I) < 4 kcal/mol). These results support a view of the apparent two-state protein folding reaction surface as a fast pre-equilibrium between the denatured state and a series of high-energy species. The net folding rate is a product of the equilibrium constant of the highest-energy species and a transmission rate. For ubiquitin, we estimate the transmission rate to be approximately 10(4) s-1. Implications for the role of unfolded chain diffusion on folding rates and barrier heights are discussed.

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