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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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.
About this article
Journal of Biological Physics (2012)