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Construction of a Family of Cys2His2 Zinc Binding Sites in the Hydrophobic Core of Thioredoxin by Structure-Based Design

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Department of Biochemistry, Box 3711, Duke University Medical Center, Durham, North Carolina 27710
Cite this: Biochemistry 1998, 37, 23, 8269–8277
Publication Date (Web):May 21, 1998
Copyright © 1998 American Chemical Society

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    A semi-automated, rational design strategy has been used to introduce a family of seven single, mononuclear Cys2His2 zinc sites at various locations in the hydrophobic core of Escherichia coli thioredoxin, a protein that is normally devoid of metal centers. The electronic absorption spectra of the CoII complexes show that five of these designed proteins bind metal with the intended tetrahedral geometry. The designed sites differ in their metal-binding constants and effects on protein stability. Since these designs are constructed within the same host protein framework, comparison of their behavior allows a qualitative evaluation of dominant factors that contribute to metal-binding and metal-mediated protein stabilization. Metal-binding constants are dominated by steric interactions between the buried, designed coordination sphere and the surrounding protein matrix. Metal-mediated stability is the consequence of differential binding to the native and unfolded states. Increased interactions with the unfolded state decrease the stabilizing effect of metal binding. The affinity for the unfolded state is dependent on the placement of the primary coordination sphere residues within the linear protein sequence. These results indicate that a protein fold can have a remarkably broad potential for accommodating metal-mediated cross-links and suggest strategies for engineering protein stability by constructing metal sites that maximize metal binding to the native state and minimize binding to the unfolded state.

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     This work was supported by a grant from the National Institutes of Health R29GM49871 to H.W.H. M.S.W. and C.Z.G. acknowledge support from a training grant from the National Institutes of Health 5T32-GM-08487-04.


     To whom correspondence should be addressed. Phone:  (919)-681-5885. Fax:  (919)-684-8885. E-mail:  hellinga@

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