Gradual folding of an off-pathway molten globule detected at the single-molecule level

S. Lindhoud, M. Pirchi, A.H. Westphal, G. Haran*, C.P.M. van Mierlo*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

15 Citations (Scopus)


Molten globules (MGs) are compact, partially folded intermediates that are transiently present during folding of many proteins. These intermediates reside on or off the folding pathway to native protein. Conformational evolution during folding of off-pathway MGs is largely unexplored. Here, we characterize the denaturant-dependent structure of apoflavodoxin's off-pathway MG. Using single-molecule fluorescence resonance energy transfer (smFRET), we follow conversion of unfolded species into MG down to denaturant concentrations that favor formation of native protein. Under strongly denaturing conditions, fluorescence resonance energy transfer histograms show a single peak, arising from unfolded protein. The smFRET efficiency distribution shifts to higher value upon decreasing denaturant concentration because the MG folds. Strikingly, upon approaching native conditions, the fluorescence resonance energy transfer efficiency of the MG rises above that of native protein. Thus, smFRET exposes the misfolded nature of apoflavodoxin's off-pathway MG. We show that conversion of unfolded into MG protein is a gradual, second-order-like process that simultaneously involves separate regions within the polypeptide.
Original languageEnglish
Pages (from-to)3148-3157
JournalJournal of Molecular Biology
Issue number19
Publication statusPublished - 2015


  • Alexa Fluor
  • folding intermediate
  • protein misfolding
  • second-order-like transition
  • single-molecule FRET


Dive into the research topics of 'Gradual folding of an off-pathway molten globule detected at the single-molecule level'. Together they form a unique fingerprint.

Cite this