Circumventing the stability-function trade-off in an engineered FN3 domain

Benjamin T. Porebski, Paul J. Conroy, Nyssa Drinkwater, Peter Schofield, Rodrigo Vazquez-Lombardi, Morag R. Hunter, David E. Hoke, Daniel Christ, Sheena McGowan, Ashley M. Buckle

Research output: Contribution to journalArticleResearchpeer-review

14 Citations (Scopus)


The favorable biophysical attributes of non-antibody scaffolds make them attractive alternatives to monoclonal antibodies. However, due to the well-known stability-function trade-off, these gains tend to be marginal after functional selection. A notable example is the fibronectin Type III (FN3) domain, FNfn10, which has been previously evolved to bind lysozyme with 1 pM affinity (FNfn10-α-lys), but suffers from poor thermodynamic and kinetic stability. To explore this stability-function compromise further, we grafted the lysozyme-binding loops from FNfn10-α-lys onto our previously engineered, ultra-stable FN3 scaffold, FN3con. The resulting variant (FN3con-α-lys) bound lysozyme with a markedly reduced affinity, but retained high levels of thermal stability. The crystal structure of FNfn10-α-lys in complex with lysozyme revealed unanticipated interactions at the protein-protein interface involving framework residues of FNfn10-α-lys, thus explaining the failure to transfer binding via loop grafting. Utilizing this structural information, we redesigned FN3con-α-lys and restored picomolar binding affinity to lysozyme, while maintaining thermodynamic stability (with a thermal melting temperature 2-fold higher than that of FNfn10-α-lys). FN3con therefore provides an exceptional window of stability to tolerate deleterious mutations, resulting in a substantial advantage for functional design. This study emphasizes the utility of consensus design for the generation of highly stable scaffolds for downstream protein engineering studies.
Original languageEnglish
Pages (from-to)541-550
Number of pages9
JournalProtein Engineering Design and Selection
Issue number11
Publication statusPublished - 1 Nov 2016


  • consensus design
  • loop grafting
  • protein engineering
  • stability-function trade-off
  • X-ray crystallography

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