Residue-resolved stability of full-consensus ankyrin repeat proteins probed by NMR.
We investigated the stability determinants and the unfolding characteristics of full-consensus designed ankyrin repeat proteins (DARPins) by NMR. Despite the repeating sequence motifs, the resonances could be fully assigned using (2)H,(15)N,(13)C triple-labeled proteins. To remove further ambiguities, we attached paramagnetic spin labels to either end of these elongated proteins, which attenuate the resonances of the spatially closest residues. Deuterium exchange experiments of DARPins with two and three internal repeats between N- and C-terminal capping repeats (NI(2)C, NI(3)C) and NI(3)C_Mut5, where the C-cap had been reengineered, indicate that the stability of the full-consensus ankyrin repeat proteins is strongly dependent on the coupling between repeats, as the stabilized cap decreases the exchange rate throughout the whole protein. Some amide protons require more than a year to exchange at 37 degrees C, highlighting the extraordinary stability of the proteins. Denaturant-induced unfolding, followed by deuterium exchange, chemical shift change, and heteronuclear nuclear Overhauser effects, is consistent with an Ising-type description of equilibrium folding for NI(3)C_Mut5, while for native-state deuterium exchange, we postulate local fluctuations to dominate exchange as unfolding events are too slow in these very stable proteins. The location of extraordinarily slowly exchanging protons indicates a very stable core structure in the DARPins that combines hydrophobic shielding with favorable electrostatic interactions. These investigations help the understanding of repeat protein architecture and the further design of DARPins for biomedical applications where high stability is required.
Wetzel
Svava K
SK
Institute of Biochemistry, University of Zürich, CH-8057 Zürich, Switzerland.
Ewald
Christina
C
Settanni
Giovanni
G
Jurt
Simon
S
Plückthun
Andreas
A
Zerbe
Oliver
O
D017089:Ankyrin Repeat; D017487:Ankyrins / Q000737:chemistry*; D002942:Circular Dichroism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013113:Spin Labels
D017487:Ankyrins; D013113:Spin Labels