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

Casbarra, A; Birolo, L; Infusini, G; DAL Piaz, F; Svensson, M; Pucci, P; Svanborg, Catharina LU and Marino, G (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)
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author
organization
publishing date
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
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
2007-10-17 16:15:08
date last changed
2017-11-19 03:35:37
@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},
  keyword      = {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},
  volume       = {13},
  year         = {2004},
}