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⇱ Engineered metal binding sites map the heterogeneous folding landscape of a coiled coil | Nature Structural & Molecular Biology

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Abstract

To address whether proteins fold along multiple pathways, i,i+4 bi-histidine metal binding sites are introduced into dimeric and crosslinked versions of the leucine zipper region of the growth control transcription factor, GCN4. Divalent metal ion binding enhances both the equilibrium and folding activation free energies for GCN4. The enhancement of folding rates quantifies the fraction of molecules that have the binding site in a helical geometry in the transition state. Hence, this new method, termed Ξ¨-analysis, identifies the degree of pathway heterogeneity for a protein that folds in a two-state manner, a capability that is generally unavailable even with single molecule methods. Adjusting metal ion concentration continuously varies the stability of the bi-histidine region without additional structural perturbation to the protein. For dimeric and crosslinked versions, the accompanying changes in kinetic barrier heights at each metal ion concentration maps the folding landscape as well as establishes the importance of connectivity in pathway selection. Furthermore, this method can be generalized to other biophysical studies, where the ability to continuously tune the stability of a particular region with no extraneous structural perturbation is advantageous.

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Figure 1: Designed metal ion binding variants of the coiled coil.
Figure 2: Denaturant dependence of folding at different metal concentrations.
Figure 3: Determining folding models.

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Acknowledgements

We thank S. Meredith, X. Fang, T. Pan, N. Kallenbach, S.W. Englander, D. Baker, A. FernΓ‘ndez, A. Kossiakoff and our group members for numerous enlightening discussions. We also thank G. Reddy for peptide synthesis supported by a grant from the National Cancer Institute. This work was supported by grants from the National Institutes of Health and The Packard Foundation Interdisciplinary Science Program (T.R.S., P. Thiyagarajan, S. Berry, D. Lynn and S. Meredith).

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Authors and Affiliations

  1. Department of Biochemistry & Molecular Biology, University of Chicago, Chicago, 60637, Illinois, USA

    Bryan A. Krantz & Tobin R. Sosnick

  2. Institute for Biophysical Dynamics, University of Chicago, Chicago, 60637, Illinois, USA

    Tobin R. Sosnick

Authors
  1. Bryan A. Krantz
  2. Tobin R. Sosnick

Corresponding author

Correspondence to Tobin R. Sosnick.

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Krantz, B., Sosnick, T. Engineered metal binding sites map the heterogeneous folding landscape of a coiled coil. Nat Struct Mol Biol 8, 1042–1047 (2001). https://doi.org/10.1038/nsb723

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