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

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Author

  • A Casbarra
  • L Birolo
  • G Infusini
  • F DAL Piaz
  • M Svensson
  • P Pucci
  • Catharina Svanborg
  • G Marino

Summary, in English

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.

Publishing year

2004

Language

English

Pages

1322-1330

Publication/Series

Protein Science

Volume

13

Issue

5

Document type

Journal article

Publisher

The Protein Society

Topic

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

Keywords

  • limited proteolysis
  • H/D exchange
  • conformational analysis
  • HAMLET
  • alpha-lactalbumin

Status

Published

ISBN/ISSN/Other

  • ISSN: 1469-896X