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Protein Science (2004), 13:1572-1585. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Modulation of the structural integrity of helix F in apomyoglobin by single amino acid replacements

Paola Picotti, Anna Marabotti1, Alessandro Negro, Valeria Musi, Barbara Spolaore, Marcello Zambonin and Angelo Fontana

CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, I-35121 Padua, Italy

(RECEIVED January 19, 2004; FINAL REVISION March 8, 2004; ACCEPTED March 9, 2004)



Abstract

The conformational features of native and mutant forms of sperm-whale apomyoglobin (apoMb) at neutral pH were probed by limited proteolysis experiments utilizing up to eight proteases of different substrate specificities. It was shown that all proteases selectively cleave apoMb at the level of chain segment 82–94 (HEAELKPLAQSHA), encompassing helix F in the X-ray structure of the holo form of the native protein; for example, thermolysin cleaves the Pro 88–Leu 89 peptide bond. These results indicate that helix F is highly flexible or largely disrupted in apoMb. Because helix F contains the helix-breaking Pro 88 residue, we propose that helix F is kept in place in the native holo protein by a variety of helix–heme stabilizing interactions. To modulate the stability of helix F, the Pro88Ala and Pro88Gly mutants were prepared by site-directed mutagenesis, and their conformational properties investigated by both far-UV circular dichroism spectroscopy and limited proteolysis. The helix content of the Pro88Ala mutant was somewhat enhanced with respect to that of both native and Pro88Gly mutant, as expected from the fact that Ala is the strongest helix inducer among the 20 amino acid residues. The rate of limited proteolysis of the three apoMb variants by thermolysin and proteinase K was in the order native > Pro88Gly >> Pro88Ala, in agreement with the scale of helix propensity of Ala, Gly, and Pro. The possible role of the flexible/unfolded chain segment 82–94 for the function and fate of apoMb at the cellular level is discussed.

Keywords: apomyoglobin; circular dichroism; limited proteolysis; mass spectrometry; protein dynamics; protein engineering

Abbreviations: CD, circular dichroism • apoMb, apomyoglobin, that is, heme-free myoglobin • P88G and P88A, mutant apoMb with Pro substituted with Gly or Ala, respectively, at position 88 of the 153-residue chain of the protein • MG, molten globule • 3D, three-dimensional • EDTA, ethylenedi-aminetetraacetic acid • HPLC, high-performance liquid chromatography • NMR, nuclear magnetic resonance • TFA, trifluoroacetic acid • Tris, tris(hydroxymethyl)aminomethane • E:S, enzyme to substrate ratio • K, proteinase K • Su, subtilisin • Th, thermolysin • Ch, chymotrypsin • V8, protease V8 from S. aureus • T, trypsin • P, papain • E, elastase • [{theta}], mean residue ellipticity • PAGE, polyacrylamide gel electrophoresis • SDS, sodium dodecyl sulfate • RP, reverse-phase • UV, ultraviolet • MS, mass spectrometry • ESI, electrosprayionization.

Reprint requests to: Angelo Fontana, CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, I-35121 Padua, Italy; e-mail: angelo.fontana{at}unipd.it; fax: 39-049-8276159.

1 Present address: Laboratory of Bioinformatics, Institute of Food Science, National Council of Research, Avellino, Italy.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04635304.

Supplemental material: see www.proteinscience.org

2 In this study, helix F in the holo form of sperm-whale myoglobin is considered to comprise residues 82–94, at variance from chain segment 86–94 of other studies (e.g. Lecomte et al. 1996, 1999). However, the exact location of amino- and carboxyterminal residues in helical segments in protein structures cannot be unequivocally defined. In the X-ray structure of the highly homologous horse myoglobin, helix F comprises residues 82–97 (Evans and Brayer 1990).

3 This work was presented at the Fifth European Symposium of the Protein Society, Florence, Italy, March 29–April 2, 2003. (Protein Sci. Suppl. 1 : Commun. 118.) formed. Identification of these fragments enabled us to locate the sites of limited proteolysis along the 153-residue chain of apoMb.


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