Modulation of the structural integrity of helix F in apomyoglobin by single amino acid replacements

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

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)1572-1585
Number of pages14
JournalProtein Science
Volume13
Issue number6
DOIs
Publication statusPublished - Jun 2004

Keywords

  • Apomyoglobin
  • Circular dichroism
  • Limited proteolysis
  • Mass spectrometry
  • Protein dynamics
  • Protein engineering

ASJC Scopus subject areas

  • Biochemistry

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