Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis

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Abstract

A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human alpha-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-alpha-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the P-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the beta-domain of a-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.

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Subject classification (UKÄ) – MANDATORY

  • Immunology in the medical area
  • Microbiology in the medical area

Keywords

  • limited proteolysis, H/D exchange, conformational analysis, HAMLET, alpha-lactalbumin
Original languageEnglish
Pages (from-to)1322-1330
JournalProtein Science
Volume13
Issue number5
Publication statusPublished - 2004
Publication categoryResearch
Peer-reviewedYes