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

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.

Birolo, L (författare)
Infusini, G (författare)
DAL Piaz, F (författare)
Svensson, M (författare)
Pucci, P (författare)
Svanborg, CatharinaLund University,Lunds universitet,Avdelningen för mikrobiologi, immunologi och glykobiologi - MIG,Institutionen för laboratoriemedicin,Medicinska fakulteten,Division of Microbiology, Immunology and Glycobiology - MIG,Department of Laboratory Medicine,Faculty of Medicine(Swepub:lu)mmb-csv (författare)
Marino, G (författare)
Avdelningen för mikrobiologi, immunologi och glykobiologi - MIGInstitutionen för laboratoriemedicin (creator_code:org_t)

Av författaren/redakt...: Casbarra, A; Birolo, L; Infusini, G; DAL Piaz, F; Svensson, M; Pucci, P; visa fler...; Svanborg, Cathar ...; Marino, G; visa färre...

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