Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis
Författare
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.
Publiceringsår
2004
Språk
Engelska
Sidor
1322-1330
Publikation/Tidskrift/Serie
Protein Science
Volym
13
Issue
5
Dokumenttyp
Artikel i tidskrift
Förlag
The Protein Society
Ämne
- Immunology in the medical area
- Microbiology in the medical area
Nyckelord
- limited proteolysis
- H/D exchange
- conformational analysis
- HAMLET
- alpha-lactalbumin
Status
Published
ISBN/ISSN/Övrigt
- ISSN: 1469-896X