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Structural Model for an Alkaline Form of Ferricytochrome c

Assfalg, Michael and Bertini, Ivano and Dolfi, Alessandra and Turano, Paola and Mauk, A. Grant and Rosell, Federico I. and Gray, Harry B. (2003) Structural Model for an Alkaline Form of Ferricytochrome c. Journal of the American Chemical Society, 125 (10). pp. 2913-2922. ISSN 0002-7863.

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An ^(15)N-enriched sample of the yeast iso-1-ferricytochrome c triple variant (Lys72Ala/Lys79Ala/Cys102Thr) in an alkaline conformation was examined by NMR spectroscopy. The mutations were planned to produce a cytochrome c with a single conformer. Despite suboptimal conditions for the collection of spectra (i.e., pH ≈ 11), NMR remains a suitable investigation technique capable of taking advantage of paramagnetism. 76% of amino acids and 49% of protons were assigned successfully. The assignment was in part achieved through standard methods, in part through the identification of groups maintaining the same conformation as in the native protein at pH 7 and, for a few other residues, through a tentative analysis of internuclear distance predictions. Lys73 was assigned as the axial ligand together with His18. In this manner, 838 meaningful NOEs for 108 amino acids, 50 backbone angle constraints, and 203 pseudocontact shifts permitted the convergence of randomly generated structures to a family of conformers with a backbone RMSD of 1.5 ± 0.2 Å. Most of the native cytochrome c conformation is maintained at high pH. The NOE pattern that involves His18 clearly indicates that the proximal side of the protein, including the 20s and 40s loops, remains essentially intact. Structural differences are concentrated in the 70−80 loop, because of the replacement of Met80 by Lys73 as an axial ligand, and in the 50s helix facing that loop; as a consequence, there is increased exposure of the heme group to solvent. Based on several spectral features, we conclude that the folded polypeptide is highly fluxional.

Item Type:Article
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URLURL TypeDescription Information
Assfalg, Michael0000-0001-9331-3169
Turano, Paola0000-0002-7683-8614
Gray, Harry B.0000-0002-7937-7876
Additional Information:© 2003 American Chemical Society. Received 5 June 2002. Published online 14 February 2003. Published in print 1 March 2003. We thank the MIUR COFIN2001 and EU TMR Network (FMRX-CT98-0218) (I.B.), Italian CNR (Progetto Finalizzato Biotecnologie 01.00359.PF49) (P.T.), Operating Grant MT-14021 from the Canadian Institutes of Health Research and a Canada Research Chair (A.G.M.), and the National Science Foundation (H.B.G.).
Funding AgencyGrant Number
Ministero dell'Istruzione, dell'Università della Ricerca (MIUR)COFIN2001
European Union TMR NetworkFMRX-CT98-0218
Canadian Institutes of Health ResearchMT-14021
Canada Research Chairs ProgramUNSPECIFIED
Consiglio Nazionale delle Ricerche (CNR)UNSPECIFIED
Issue or Number:10
Record Number:CaltechAUTHORS:20170414-134611221
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Official Citation:Structural Model for an Alkaline Form of Ferricytochrome c Michael Assfalg, Ivano Bertini, Alessandra Dolfi, Paola Turano, A. Grant Mauk, Federico I. Rosell, and Harry B. Gray Journal of the American Chemical Society 2003 125 (10), 2913-2922 DOI: 10.1021/ja027180s
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:76575
Deposited By: Ruth Sustaita
Deposited On:14 Apr 2017 22:38
Last Modified:22 Nov 2019 09:58

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