Slow formation of aggregation-resistant β-sheet folding intermediates

Abstract

Protein folding has been studied extensively for decades, yet our ability to predict how proteins reach their native state from a mechanistic perspective is still rudimentary at best, limiting our understanding of folding-related processes in vivo and our ability to manipulate proteins in vitro. Here, we investigate the in vitro refolding mechanism of a large β-helix protein, pertactin, which has an extended, elongated shape. At 55 kDa, this single domain, all-β-sheet protein allows detailed analysis of the formation of β-sheet structure in larger proteins. Using a combination of fluorescence and far-UV circular dichroism spectroscopy, we show that the pertactin β-helix refolds remarkably slowly, with multiexponential kinetics. Surprisingly, despite the slow refolding rates, large size, and β-sheet-rich topology, pertactin refolding is reversible and not complicated by off-pathway aggregation. The slow pertactin refolding rate is not limited by proline isomerization, and 30% of secondary structure formation occurs within the rate-limiting step. Furthermore, site-specific labeling experiments indicate that the β-helix refolds in a multistep but concerted process involving the entire protein, rather than via initial formation of the stable core substructure observed in equilibrium titrations. Hence pertactin provides a valuable system for studying the refolding properties of larger, β-sheet-rich proteins, and raises intriguing questions regarding the prevention of aggregation during the prolonged population of partially folded, β-sheet-rich refolding intermediates. © 2009 Wiley-Liss, Inc.