Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis |
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Authors: | Casbarra Annarita Birolo Leila Infusini Giuseppe Dal Piaz Fabrizio Svensson Malin Pucci Piero Svanborg Catharina Marino Gennaro |
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Institution: | Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, I-80126 Napoli, Italy. |
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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 beta-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 alpha-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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Keywords: | HAMLET α-lactalbumin conformational analysis H/D exchange limited proteolysis |
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