De novo folding of GFP fusion proteins: High efficiency in eukaryotes but not in bacteria

Hung Chun Chang, Christian M. Kaiser, F. Ulrich Hartl, José M. Barral

Research output: Contribution to journalArticlepeer-review

74 Scopus citations

Abstract

Eukaryotic genomes encode a considerably higher fraction of multi-domain proteins than their prokaryotic counterparts. It has been postulated that efficient co-translational and sequential domain folding has facilitated the explosive evolution of multi-domain proteins in eukaryotes by the recombination of pre-existent domains. Here, we tested whether eukaryotes and bacteria differ generally in the folding efficiency of multi-domain proteins generated by domain recombination. To this end, we compared the folding behavior of a series of recombinant proteins comprised of green fluorescent protein (GFP) fused to four different robustly folding proteins through six different linkers upon expression in Escherichia coli and the yeast Saccharomyces cerevisiae. We found that, unlike yeast, bacteria are remarkably inefficient at folding these fusion proteins, even at comparable levels of expression. In vitro and in vivo folding experiments demonstrate that the GFP domain imposes significant constraints on de novo folding of its fusion partners in bacteria, consistent with a largely post-translational folding mechanism. This behavior may result from an interference of GFP with adjacent domains during folding due to the particular topology of the β-barrel GFP structure. By following the accumulation of enzymatic activity, we found that the rate of appearance of correctly folded fusion protein per ribosome is indeed considerably higher in yeast than in bacteria.

Original languageEnglish (US)
Pages (from-to)397-409
Number of pages13
JournalJournal of Molecular Biology
Volume353
Issue number2
DOIs
StatePublished - Oct 21 2005
Externally publishedYes

Keywords

  • E. coli
  • GFP
  • Multi-domain proteins
  • Protein folding
  • S. cerevisiae

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Structural Biology

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