Slow formation of aggregation-resistant beta-sheet folding intermediates.
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 beta-helix protein, pertactin, which has an extended, elongated shape. At 55 kDa, this single domain, all-beta-sheet protein allows detailed analysis of the formation of beta-sheet structure in larger proteins. Using a combination of fluorescence and far-UV circular dichroism spectroscopy, we show that the pertactin beta-helix refolds remarkably slowly, with multiexponential kinetics. Surprisingly, despite the slow refolding rates, large size, and beta-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 beta-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, beta-sheet-rich proteins, and raises intriguing questions regarding the prevention of aggregation during the prolonged population of partially folded, beta-sheet-rich refolding intermediates. Proteins 2010. (c) 2009 Wiley-Liss, Inc.
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556-5670, USA.
Mirco
M
Junker
Patricia L
PL
Clark
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D007700:Kinetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D010566:Virulence Factors, Bordetella / Q000737:chemistry*
D001425:Bacterial Outer Membrane Proteins; D010566:Virulence Factors, Bordetella; C068929:pertactin; D014364:Tryptophan