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

Journal of Molecular Biology
Svava K WetzelOliver Zerbe

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 (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 pos...Continue Reading

References

Jun 1, 1994·Nature Structural Biology·V Muñoz, L Serrano
Sep 1, 1993·Proteins·Y BaiS W Englander
Sep 1, 1996·Protein Science : a Publication of the Protein Society·J A Boice, R Fairman
Oct 8, 1998·Journal of Magnetic Resonance·S A McCallumG S Rule
Aug 4, 1999·Trends in Biochemical Sciences·S G Sedgwick, S J Smerdon
Sep 12, 2001·Journal of Structural Biology·M A AndradeC P Ponting
Sep 25, 2002·Journal of Molecular Biology·Michael A LietzowPeter E Wright
Dec 4, 2002·Proceedings of the National Academy of Sciences of the United States of America·Leila K MosaviZheng-Yu Peng
Jan 9, 2003·Structure·Kit S TangLaura S Itzhaki
Feb 5, 2003·Proceedings of the National Academy of Sciences of the United States of America·Andreas KohlMarkus G Grütter
Apr 21, 2004·Nature Biotechnology·H Kaspar BinzAndreas Plückthun
Jul 31, 2004·Methods : a Companion to Methods in Enzymology·Mallela M G KrishnaS Walter Englander
Sep 21, 2004·Proceedings of the National Academy of Sciences of the United States of America·Cecilia C Mello, Doug Barrick
Apr 13, 2005·Proceedings of the National Academy of Sciences of the United States of America·Ewan R G MainLynne Regan
Jul 21, 2005·Journal of the American Chemical Society·Tommi KajanderLynne Regan
Dec 25, 2007·Journal of Molecular Biology·Tobias MerzPeer R E Mittl
Jan 1, 2008·Journal of Molecular Biology·Svava K WetzelAndreas Plückthun
May 9, 2008·Journal of Molecular Biology·Aitziber L CortajarenaLynne Regan
Dec 9, 2008·Journal of the American Chemical Society·Hagen HofmannRenate Ulbrich-Hofmann
Jul 1, 1995·Journal of Biomolecular NMR·C BartelsK Wüthrich

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Citations

Feb 23, 2013·Proceedings of the National Academy of Sciences of the United States of America·Birgit DreierAndreas Plückthun
Dec 14, 2011·Current Opinion in Structural Biology·Emily H Rubinson, Brandt F Eichman
Sep 21, 2010·Journal of Molecular Biology·Michaela A KramerMarkus G Grütter
Jul 7, 2012·Protein Science : a Publication of the Protein Society·Pietro AlfaranoAmedeo Caflisch
Dec 16, 2011·Protein Science : a Publication of the Protein Society·J J PhillipsE R G Main
Aug 19, 2014·Protein Science : a Publication of the Protein Society·Christian ReichenPeer R E Mittl
Jan 1, 2013·Journal of Molecular Biology·Carla F CervantesElizabeth A Komives
Jul 1, 2011·Current Opinion in Biotechnology·Ykelien L Boersma, Andreas Plückthun
Jan 7, 2015·Annual Review of Pharmacology and Toxicology·Andreas Plückthun
Mar 7, 2018·Proceedings of the National Academy of Sciences of the United States of America·Yuxi LiuTodd O Yeates
Jul 2, 2020·Antibodies·Adinarayana KunamneniRavi Durvasula
May 8, 2018·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·Albert Perez-RibaLaura S Itzhaki
Oct 30, 2019·Scientific Reports·Albert Perez-RibaLaura S Itzhaki
Jan 10, 2020·Current Opinion in Structural Biology·Peer Re MittlAndreas Plückthun
Aug 28, 2018·ACS Synthetic Biology·Mohammad ElGamacyAndrei N Lupas

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