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Atom-by-atom analysis of global downhill protein folding

Nature volume 442, pages 317–321 (2006)Cite this article

Abstract

Protein folding is an inherently complex process involving coordination of the intricate networks of weak interactions that stabilize native three-dimensional structures. In the conventional paradigm, simple protein structures are assumed to fold in an all-or-none process1 that is inaccessible to experiment. Existing experimental methods therefore probe folding mechanisms indirectly. A widely used approach interprets changes in protein stability2 and/or folding kinetics3,4, induced by engineered mutations, in terms of the structure of the native protein. In addition to limitations in connecting energetics with structure5, mutational methods have significant experimental uncertainties6 and are unable to map complex networks of interactions. In contrast, analytical theory predicts small barriers to folding and the possibility of downhill folding7,8. These theoretical predictions have been confirmed experimentally in recent years9,10,11, including the observation of global downhill folding12. However, a key remaining question is whether downhill folding can indeed lead to the high-resolution analysis of protein folding processes13. Here we show, with the use of nuclear magnetic resonance (NMR), that the downhill protein BBL from Escherichia coli unfolds atom by atom starting from a defined three-dimensional structure. Thermal unfolding data on 158 backbone and side-chain protons out of a total of 204 provide a detailed view of the structural events during folding. This view confirms the statistical nature of folding, and exposes the interplay between hydrogen bonding, hydrophobic forces, backbone conformation and side-chain entropy. From the data we also obtain a map of the interaction network in this protein, which reveals the source of folding cooperativity. Our approach can be extended to other proteins with marginal barriers (less than 3RT), providing a new tool for the study of protein folding.

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Figure 1: Structure and dynamics of the native ensemble.
Figure 2: Protein thermal unfolding atom by atom.
Figure 3: Mapping thermal stability on the 3D structure.
Figure 4: Interaction matrices and the emergence of folding cooperativity.

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Acknowledgements

We thank E. de Alba for help and guidance in the calculation of NMR structures with Xplor-NIH and for generating the table of structure statistics; P. Vasos for assistance with the NMR relaxation experiments; and Y. Hathout for mass-spectrometric analysis. The research described in this article was supported by the NIH and the NSF. Author Contributions D.F. performed and analysed the NMR relaxation experiments and contributed to manuscript preparation. M.S. performed all other NMR experiments and circular dichroism experiments, assigned all the NMR spectra, and contributed to manuscript preparation. V.M. conceived the project, supervised the work, developed the methods for analysis, analysed the experimental data, and contributed to manuscript preparation. The structural analysis was performed in conjunction by M.S. and V.M.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, and Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland, 20742, USA

    Mourad Sadqi, David Fushman & Victor Muñoz

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  1. Mourad Sadqi
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  2. David Fushman
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  3. Victor Muñoz
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Corresponding author

Correspondence to Victor Muñoz.

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Competing interests

The atomic coordinates of Naf-BBL have been deposited in the Protein Data Bank with the accession number 2QYU. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This file contains six sections. The first section of the contains a description of the determination of the NMR structure at 278 K, including a Supplementary Table with the structure statistics. The second section contains a more detailed description of the NMR relaxation experiments. The third section includes additional technical information about the analysis of chemical shift versus temperature curves in terms of atomic folding-unfolding equilibria. The fourth section contains the equations and formalism for calculation of the mean thermodynamic coupling index (MTCI) and matrix of residue-residue mean thermodynamic couplings from chemical shift versus temperature unfolding curves. The fifth section contains a technical description of the methods employed in this work.The sixth section is a table of the chemical shifts versus temperature for the 158 protons of the protein BBL that have been employed in this work. (DOC 5586 kb)

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Sadqi, M., Fushman, D. & Muñoz, V. Atom-by-atom analysis of global downhill protein folding. Nature 442, 317–321 (2006). https://doi.org/10.1038/nature04859

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