Calcium Binding and Disulfide Bonds Regulate the Stability of Secretagogin towards Thermal and Urea Denaturation.
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.
Department of Biochemistry and Structural Biology, Lund University, Lund, Sweden.
Kalyani
http://orcid.org/0000-0002-8790-9393
K
Sanagavarapu
Department of Biochemistry and Structural Biology, Lund University, Lund, Sweden.
Tanja
T
Weiffert
School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Dublin, Ireland.
Niamh
N
Ní Mhurchú
School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Dublin, Ireland.
David
D
O'Connell
Department of Biochemistry and Structural Biology, Lund University, Lund, Sweden.
Sara
S
Linse
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002942:Circular Dichroism; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism*; D020832:EF Hand Motifs / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006801:Humans; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D051381:Rats; D064046:Secretagogins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000378:metabolism*
D004220:Disulfides; C576556:SCGN protein, human; D064046:Secretagogins; D014508:Urea; D002118:Calcium