Projects per year
Abstract
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 language | English |
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Pages (from-to) | 541-550 |
Number of pages | 9 |
Journal | Protein Engineering Design and Selection |
Volume | 29 |
Issue number | 11 |
DOIs | |
Publication status | Published - 1 Nov 2016 |
Keywords
- consensus design
- loop grafting
- protein engineering
- stability-function trade-off
- X-ray crystallography
Projects
- 1 Finished
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Design and engineering of proteins for biotechnology and biomedicine
Buckle, A. (Primary Chief Investigator (PCI))
ARC - Australian Research Council, Monash University
1/01/15 → 24/12/18
Project: Research
Equipment
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Monash Macromolecular Crystallisation Platform (MMCP)
Kong, G. (Operator)
Faculty of Medicine Nursing and Health Sciences Research PlatformsFacility/equipment: Facility