Kinetic analysis of ribosome-bound fluorescent proteins reveals an early, stable, cotranslational folding intermediate

Devaki A. Kelkar, Amardeep Khushoo, Zhongying Yang, William R. Skach

Research output: Contribution to journalArticlepeer-review

40 Scopus citations


Protein folding in cells reflects a delicate interplay between biophysical properties of the nascent polypeptide, the vectorial nature and rate of translation, molecular crowding, and cellular biosynthetic machinery. To better understand how this complex environment affects de novo folding pathways as they occur in the cell, we expressed β-barrel fluorescent proteins derived from GFP and RFP in an in vitro system that allows direct analysis of cotranslational folding intermediates. Quantitative analysis of ribosome-bound eCFP and mCherry fusion proteins revealed that productive folding exhibits a sharp threshold as the length of polypeptide from the C terminus to the ribosome peptidyltransferase center is increased. Fluorescence spectroscopy, urea denaturation, and limited protease digestion confirmed that sequestration of only 10-15 C-terminal residues within the ribosome exit tunnel effectively prevents stable barrel formation, whereas folding occurs unimpeded when the C terminus is extended beyond the ribosome exit site. Nascent FPs with 10 of the 11 β-strands outside the ribosome exit tunnel acquire a non-native conformation that is remarkably stable in diverse environments. Upon ribosome release, these structural intermediates fold efficiently with kinetics that are unaffected by the cytosolic crowding or cellular chaperones. Our results indicate that during synthesis, fluorescent protein folding is initiated cotranslationally via rapid formation of a highly stable, on-pathway structural intermediate and that the rate-limiting step of folding involves autonomous incorporation of the 11th β-strand into the mature barrel structure.

Original languageEnglish (US)
Pages (from-to)2568-2578
Number of pages11
JournalJournal of Biological Chemistry
Issue number4
StatePublished - Jan 20 2012

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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