SDS-PAGE analysis of secretagogin dimers.

<p>(3A) Non-reducing gel of apo secretagogin. (3B) Non-reducing gel of calcium-loaded secretagogin. Lane 1 has pre stained protein ladder. Lanes 2–12 have 1 mg/mL secretagogin (30 μM) with 20 mM DTT, 10 mM DTT, 7 mM DTT, 5 mM DTT, 4 mM DTT, 3 mM DTT, 2 mM DTT, 1 mM DTT, 0.7 mM DTT (not present in 3B), 0.5 mM DTT and no DTT respectively. M = monomer; D = dimer; T = trimer (3C) Percent of monomer versus concentrations of DTT and dimer versus DTT concentration. Red: apo protein; blue: calcium-loaded protein; filled box: monomer; non-filled box: dimer.</p

Urea induced denaturation of secretagogin in non-reducing conditions (10 mM Tris, 0.15 M KCl [A, D] with 1 mM CaCl₂ or 0.5 mM EDTA. pH: 7.5) and reducing conditions (10 mM Tris, 0.15 M KCl, 1mM [B, E] or 4 mM DTT [C, F] with 1 mM CaCl₂ or 0.5 mM EDTA, pH: 7.5).

<p>Blue and red curves represent calcium-loaded and apo form of secretagogin, respectively. [Θ]_{222 nm} is the ellipticity at 222 nm; λ max is the wavelength at which the fluorescent intensity is highest; Cm is the urea concentration at the transition midpoint. In panels a-c are shown [Θ]_{222 nm} versus urea concentration and in panels d-f λ max versus urea concentration.</p

The reversibility ranges of secretagogin analyzed by circular dichroism spectroscopy.

<p>Curves here represent temperature denaturation of both apo and calcium-loaded secretagogin during heating from 20–55°C and 20–65°C and from the maximum temperature to 20°C, for apo and calcium-loaded secretagogin at 0, 1 and 4 M urea and 0, 1 and 4 mM DTT. [Θ]_{222 nm} is ellipticity at 222 nm. Blue and pale blue dots represent calcium-loaded secretagogin during heating from 20°C to test temperature and reverse scan to 20°C, respectively. Red and pale red dots represent apo secretagogin during heating from 20°C to test temperature and reverse scan to 20°C, respectively. (A) [Θ]_{222 nm} versus temperature and total absorbance (nm) versus temperature at 20–55°C. (B) [Θ]_{222 nm} versus temperature and total absorbance (nm) versus temperature obtained at 20–65°C.</p

Cartoon representing the free energy changes (ΔG) involved in folding and calcium binding of an EF-hand protein.

<p>The total free energy change from its unfolded state to calcium-loaded state includes contributions from both folding and binding. Blue circles represent Ca²⁺ ions.</p

Various buffer conditions used to study stability of secretagogin.

<p>Various buffer conditions used to study stability of secretagogin.</p

Calcium Binding and Disulfide Bonds Regulate the Stability of Secretagogin towards Thermal and Urea Denaturation - Fig 6

<p><i>Lane</i> 1: Apo secretagogin reduced in 20 mM DTT and dialyzed in glutathione redox buffer of -170 mV potential. Lane 2: Apo secretagogin reduced in 20 mM DTT.</p

Illustration of pairwise Cys-Cys distance in human secretagogin structure mapped on <i>Dan rerio</i> secretagogin X-ray structure, PDB ID: 2BE4.

<p>Illustration of pairwise Cys-Cys distance in human secretagogin structure mapped on <i>Dan rerio</i> secretagogin X-ray structure, PDB ID: 2BE4.</p

Anti secretagogin Western blot analysis of endogenous secretagogin in BRIN-BD11 insulinoma cell lysates at selected concentrations of DTT.

<p><b>(5A)</b> Non-reducing Western blot of native secretagogin exposed for 30 s with low sensitivity Pierce^™ chemiluminescent Western blotting substrate (Thermo Scientific, cat. no. 32106). (5B) Non-reducing Western blot of native secretagogin exposed for 5 min with high sensitivity BM chemiluminescent Western blotting substrate (Roche, cat. no. 11500708001). Each lane has 30 μg of total protein with 20 mM DTT, 10 mM DTT, 5 mM DTT, 1 mM DTT, and no DTT respectively. M = monomer; D = dimer.</p

Summary of apparent Tm and the real reversibility temperature range of secretagogin in various conditions studied.

<p>Summary of apparent Tm and the real reversibility temperature range of secretagogin in various conditions studied.</p

Illustration of pairwise Cys-Cys distance in human secretagogin structure mapped on <i>Dan rerio</i> secretagogin X-ray structure, PDB ID: 2BE4.

<p>Illustration of pairwise Cys-Cys distance in human secretagogin structure mapped on <i>Dan rerio</i> secretagogin X-ray structure, PDB ID: 2BE4.</p