Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis
(2004) In Protein Science 13(5). p.1322-1330- 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 P-domain... (More)
- 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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/280093
- author
- Casbarra, A ; Birolo, L ; Infusini, G ; DAL Piaz, F ; Svensson, M ; Pucci, P ; Svanborg, Catharina LU and Marino, G
- organization
- publishing date
- 2004
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- limited proteolysis, H/D exchange, conformational analysis, HAMLET, alpha-lactalbumin
- in
- Protein Science
- volume
- 13
- issue
- 5
- pages
- 1322 - 1330
- publisher
- The Protein Society
- external identifiers
-
- wos:000221042200016
- pmid:15075403
- scopus:1942441021
- pmid:15075403
- ISSN
- 1469-896X
- DOI
- 10.1110/ps.03474704
- language
- English
- LU publication?
- yes
- id
- a01fba00-e273-48b3-9682-48dab9db8ec6 (old id 280093)
- date added to LUP
- 2016-04-01 12:17:48
- date last changed
- 2022-01-27 01:39:36
@article{a01fba00-e273-48b3-9682-48dab9db8ec6, 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 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.}}, author = {{Casbarra, A and Birolo, L and Infusini, G and DAL Piaz, F and Svensson, M and Pucci, P and Svanborg, Catharina and Marino, G}}, issn = {{1469-896X}}, keywords = {{limited proteolysis; H/D exchange; conformational analysis; HAMLET; alpha-lactalbumin}}, language = {{eng}}, number = {{5}}, pages = {{1322--1330}}, publisher = {{The Protein Society}}, series = {{Protein Science}}, title = {{Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis}}, url = {{http://dx.doi.org/10.1110/ps.03474704}}, doi = {{10.1110/ps.03474704}}, volume = {{13}}, year = {{2004}}, }