Most principles of protein folding emerged from refolding studies in vitro on small, soluble proteins, because large ones tend to misfold and aggregate. We developed a folding assay allowing the study of large proteins in detergent such that the extent of cellular assistance required for proper folding can be determined. We identified a critical step in the in vivo folding pathway of influenza virus hemagglutinin (HA). Only the formation of the first few disulfides in the top domain of HA required the intact endoplasmic reticulum. After that, HA proceeded to fold efficiently in a very dilute solution, despite its size and complexity. This study paves the way for detailed structural analyses during the folding of complex proteins.
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We thank H. Tabak, A. Ora and A. Azuaga-Fortes for comments, discussions and critical reading of the manuscript, and B. Kleizen, M. Molinari, A. Helenius and members of the Braakman lab for fruitful discussions. We thank J. Smit for preparing the cartoon of HA (Fig. 2a). The work was supported by a grant from Telethon Foundation, Italy (M.C.M.), a European Union Marie Curie Research Training grant (M.C.M.), and Netherlands Organization for Scientific Research/Chemical Sciences (I.M.L. and I.B.).
The authors declare no competing financial interests.
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Maggioni, M., Liscaljet, I. & Braakman, I. A critical step in the folding of influenza virus HA determined with a novel folding assay. Nat Struct Mol Biol 12, 258–263 (2005). https://doi.org/10.1038/nsmb897