Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins

J Mol Biol. 2008 Feb 8;376(1):241-57. doi: 10.1016/j.jmb.2007.11.046. Epub 2007 Nov 22.


Full-consensus designed ankyrin repeat proteins were designed with one to six identical repeats flanked by capping repeats. These proteins express well in Escherichia coli as soluble monomers. Compared to our previously described designed ankyrin repeat protein library, randomized positions have now been fixed according to sequence statistics and structural considerations. Their stability increases with length and is even higher than that of library members, and those with more than three internal repeats are resistant to denaturation by boiling or guanidine hydrochloride. Full denaturation requires their heating in 5 M guanidine hydrochloride. The folding and unfolding kinetics of the proteins with up to three internal repeats were analyzed, as the other proteins could not be denatured. Folding is monophasic, with a rate that is nearly identical for all proteins ( approximately 400-800 s(-1)), indicating that essentially the same transition state must be crossed, possibly the folding of a single repeat. In contrast, the unfolding rate decreases by a factor of about 10(4) with increasing repeat number, directly reflecting thermodynamic stability in these extraordinarily slow denaturation rates. The number of unfolding phases also increases with repeat number. We analyzed the folding thermodynamics and kinetics both by classical two-state and three-state cooperative models and by an Ising-like model, where repeats are considered as two-state folding units that can be stabilized by interacting with their folded nearest neighbors. This Ising model globally describes both equilibrium and kinetic data very well and allows for a detailed explanation of the ankyrin repeat protein folding mechanism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Ankyrin Repeat*
  • Ankyrins / chemistry*
  • Ankyrins / metabolism*
  • Circular Dichroism
  • Escherichia coli / genetics
  • Gene Expression
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Folding*
  • Spectrometry, Fluorescence


  • Ankyrins