Extreme stability in de novo-designed repeat arrays is determined by unusually stable short-range interactions

Kathryn Geiger-Schuller, Kevin Sforza, Max Yuhas, Fabio Parmeggiani, David Baker, Doug Barrick*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

21 Citations (Scopus)
358 Downloads (Pure)


Designed helical repeats (DHRs) are modular helix–loop–helix–loop protein structures that are tandemly repeated to form a superhelical array. Structures combining tandem DHRs demonstrate a wide range of molecular geometries, many of which are not observed in nature. Understanding cooperativity of DHR proteins provides insight into the molecular origins of Rosetta-based protein design hyperstability and facilitates comparison of energy distributions in artificial and naturally occurring protein folds. Here, we use a nearest-neighbor Ising model to quantify the intrinsic and interfacial free energies of four different DHRs. We measure the folding free energies of constructs with varying numbers of internal and terminal capping repeats for four different DHR folds, using guanidine-HCl and glycerol as destabilizing and solubilizing cosolvents. One-dimensional Ising analysis of these series reveals that, although interrepeat coupling energies are within the range seen for naturally occurring repeat proteins, the individual repeats of DHR proteins are intrinsically stable. This favorable intrinsic stability, which has not been observed for naturally occurring repeat proteins, adds to stabilizing interfaces, resulting in extraordinarily high stability. Stable repeats also impart a downhill shape to the energy landscape for DHR folding. These intrinsic stability differences suggest that part of the success of Rosetta-based design results from capturing favorable local interactions.

Original languageEnglish
Article number034102
Pages (from-to)7539-7544
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number29
Early online date29 Jun 2018
Publication statusPublished - 17 Jul 2018


  • DHRs
  • Ising model
  • Protein design
  • Repeat proteins
  • Rosetta


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