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Calcium binding and disulfide bonds regulate the stability of Secretagogin towards thermal and urea denaturation

Sanagavarapu, Kalyani LU ; Weiffert, Tanja LU ; Mhurchú, Niamh Ní; O'Connell, David and Linse, Sara LU (2016) In PLoS ONE 11(11).
Abstract

Secretagogin is a calcium-sensor protein with six EF-hands. It is widely expressed in neurons and neuro-endocrine cells of a broad range of vertebrates including mammals, fishes and amphibia. The protein plays a role in secretion and interacts with several vesicle-associated proteins. In this work, we have studied the contribution of calcium binding and disulfide-bond formation to the stability of the secretagogin structure towards thermal and urea denaturation. SDS-PAGE analysis of secretagogin in reducing and non-reducing conditions identified a tendency of the protein to form dimers in a redox-dependent manner. The denaturation of apo and Calcium-loaded secretagogin was studied by circular dichroism and fluorescence spectroscopy... (More)

Secretagogin is a calcium-sensor protein with six EF-hands. It is widely expressed in neurons and neuro-endocrine cells of a broad range of vertebrates including mammals, fishes and amphibia. The protein plays a role in secretion and interacts with several vesicle-associated proteins. In this work, we have studied the contribution of calcium binding and disulfide-bond formation to the stability of the secretagogin structure towards thermal and urea denaturation. SDS-PAGE analysis of secretagogin in reducing and non-reducing conditions identified a tendency of the protein to form dimers in a redox-dependent manner. The denaturation of apo and Calcium-loaded secretagogin was studied by circular dichroism and fluorescence spectroscopy under conditions favoring monomer or dimer or a 1:1 monomer: dimer ratio. This analysis reveals significantly higher stability towards urea denaturation of Calcium-loaded secretagogin compared to the apo protein. The secondary and tertiary structure of the Calcium-loaded form is not completely denatured in the presence of 10 M urea. Reduced and Calcium-loaded secretagogin is found to refold reversibly after heating to 95°C, while both oxidized and reduced apo secretagogin is irreversibly denatured at this temperature. Thus, calcium binding greatly stabilizes the structure of secretagogin towards chemical and heat denaturation.

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organization
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publication status
published
subject
in
PLoS ONE
volume
11
issue
11
publisher
Public Library of Science
external identifiers
  • scopus:84994051886
  • wos:000386910000050
ISSN
1932-6203
DOI
10.1371/journal.pone.0165709
language
English
LU publication?
yes
id
bc5d457d-1186-4929-92d6-1f082ebfb7a3
date added to LUP
2016-11-21 09:44:04
date last changed
2017-11-19 04:34:59
@article{bc5d457d-1186-4929-92d6-1f082ebfb7a3,
  abstract     = {<p>Secretagogin is a calcium-sensor protein with six EF-hands. It is widely expressed in neurons and neuro-endocrine cells of a broad range of vertebrates including mammals, fishes and amphibia. The protein plays a role in secretion and interacts with several vesicle-associated proteins. In this work, we have studied the contribution of calcium binding and disulfide-bond formation to the stability of the secretagogin structure towards thermal and urea denaturation. SDS-PAGE analysis of secretagogin in reducing and non-reducing conditions identified a tendency of the protein to form dimers in a redox-dependent manner. The denaturation of apo and Calcium-loaded secretagogin was studied by circular dichroism and fluorescence spectroscopy under conditions favoring monomer or dimer or a 1:1 monomer: dimer ratio. This analysis reveals significantly higher stability towards urea denaturation of Calcium-loaded secretagogin compared to the apo protein. The secondary and tertiary structure of the Calcium-loaded form is not completely denatured in the presence of 10 M urea. Reduced and Calcium-loaded secretagogin is found to refold reversibly after heating to 95°C, while both oxidized and reduced apo secretagogin is irreversibly denatured at this temperature. Thus, calcium binding greatly stabilizes the structure of secretagogin towards chemical and heat denaturation.</p>},
  articleno    = {e0165709},
  author       = {Sanagavarapu, Kalyani and Weiffert, Tanja and Mhurchú, Niamh Ní and O'Connell, David and Linse, Sara},
  issn         = {1932-6203},
  language     = {eng},
  month        = {11},
  number       = {11},
  publisher    = {Public Library of Science},
  series       = {PLoS ONE},
  title        = {Calcium binding and disulfide bonds regulate the stability of Secretagogin towards thermal and urea denaturation},
  url          = {http://dx.doi.org/10.1371/journal.pone.0165709},
  volume       = {11},
  year         = {2016},
}