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Residue-resolved stability of full-consensus ankyrin repeat proteins probed by NMR


Wetzel, S K; Ewald, C; Settanni, G; Jurt, S; Plückthun, A; Zerbe, O (2010). Residue-resolved stability of full-consensus ankyrin repeat proteins probed by NMR. Journal of Molecular Biology, 402(1):241-258.

Abstract

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 1H, 15N, 13C triple labeled proteins. To remove further ambiguities, paramagnetic spin labels were attached to either end of these elongated proteins which attenuate the resonances of the spatially closest residues. Deuterium exchange experiments of DARPins with 2 and 3 internal repeats between N- and C-terminal capping repeats (NI2C, NI3C) and NI3C_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°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 NI3C_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 indicate a very stable core structure in the DARPins which 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.

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 1H, 15N, 13C triple labeled proteins. To remove further ambiguities, paramagnetic spin labels were attached to either end of these elongated proteins which attenuate the resonances of the spatially closest residues. Deuterium exchange experiments of DARPins with 2 and 3 internal repeats between N- and C-terminal capping repeats (NI2C, NI3C) and NI3C_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°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 NI3C_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 indicate a very stable core structure in the DARPins which 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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry

07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:570 Life sciences; biology
540 Chemistry
Language:English
Date:2010
Deposited On:27 Oct 2010 12:46
Last Modified:05 Apr 2016 14:14
Publisher:Elsevier
ISSN:0022-2836
Publisher DOI:https://doi.org/10.1016/j.jmb.2010.07.031
PubMed ID:20654623
Permanent URL: https://doi.org/10.5167/uzh-35602

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