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 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.
Casbarra
Annarita
A
Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, I-80126 Napoli, Italy.
Birolo
Leila
L
Infusini
Giuseppe
G
Dal Piaz
Fabrizio
F
Svensson
Malin
M
Pucci
Piero
P
Svanborg
Catharina
C
Marino
Gennaro
G
D000595:Amino Acid Sequence; D017209:Apoptosis; D006801:Humans; D007700:Kinetics; D007768:Lactalbumin / Q000032:analysis / Q000737:chemistry*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011522:Protons
D011522:Protons; D007768:Lactalbumin