Fig 1.
(A) Alignment of EF-hands of human secretagogin (SC) and the homologues calretinin (CR), reticulo calbin (RT) and calbindin (CB). The six EF-hands are labelled in different colors (red: EF1, dark red: EF2, green: EF3, pale green: EF4, cyan: EF5 and dark blue: EF6. Cysteines are boxed. (B) Crystal structure of secretagogin from Danio rerio (zebra fish), PDB ID: 2BE4. Six EF hands are colored as above. Cysteines are shown in orange, C193 is visible on the front surface, while C253 and C269 are on the back side. RT6: (X)13 = NYGEDLTKNHDEL.
Fig 2.
Illustration of pairwise Cys-Cys distance in human secretagogin structure mapped on Dan rerio secretagogin X-ray structure, PDB ID: 2BE4.
Fig 3.
SDS-PAGE analysis of secretagogin dimers.
(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.
Fig 4.
Anti secretagogin Western blot analysis of recombinant secretagogin oligomers.
Non-reducing Western blot of apo secretagogin exposed for 2 min (4A) and 5 min (4B) with Pierce™ chemiluminescent Western blotting substrate. Lane 1–11 has 2 μg of 1 mg/mL purified secretagogin 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, 0.5 mM DTT and no DTT respectively. M = monomer; D = dimer; T = trimer.
Fig 5.
Anti secretagogin Western blot analysis of endogenous secretagogin in BRIN-BD11 insulinoma cell lysates at selected concentrations of DTT.
(5A) 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.
Fig 6.
Lane 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.
Fig 7.
Urea induced denaturation of secretagogin studied 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) using (7A) circular dichroism spectroscopy and (7B) fluorescence spectroscopy.
[Θ]222 nm is the ellipticity at 222 nm; λ is the wavelength; Panel A shows [Θ]222 nm versus λ as measured using CD spectroscopy and panel B shows fluorescence intensity versus λ as measured using fluorescent spectrometer.
Fig 8.
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).
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.
Fig 9.
Thermal stability of secretagogin as analyzed by circular dichroism spectroscopy when heated from 20 to 95°C in six buffers, A-F (Table 3).
[Θ]222 nm is ellipticity at 222 nm. Blue and pale blue dots represent calcium-loaded secretagogin under heating (20–95°C) and cooling (95–20°C), respectively. Red and pale red dots represent apo secretagogin under heating (20–95°C) and cooling (95–20°C), respectively. A. [Θ]222 nm versus temperature. B. Total absorbance (nm) versus temperature.
Table 1.
Summary of apparent Tm and the real reversibility temperature range of secretagogin in various conditions studied.
Fig 10.
The reversibility ranges of secretagogin analyzed by circular dichroism spectroscopy.
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.
Fig 11.
Reversible denaturation curves of secretagogin analyzed by circular dichroism spectroscopy.
Secretagogin is tested for temperature reversible denaturation at different temperatures in six buffers, A-F (Table 3). The protein is heated up to the maximum temperature at each specific condition where it can refold. [Θ]222 nm is ellipticity at 222 nm. Blue and pale blue dots represent calcium-loaded secretagogin heated from 20°C to test temperature and reverse scan to 20°C, respectively. Red and pale red dots represent apo secretagogin heated from 20°C to test temperature and reverse scan to 20°C, respectively. A. [Θ]222 nm versus temperature. B. Total absorbance (nm) curves versus temperature.
Fig 12.
Cartoon representing the free energy changes (ΔG) involved in folding and calcium binding of an EF-hand protein.
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.
Fig 13.
Comparison of Tm values of apo and calcium-loaded form of secretagogin and other calcium binding proteins.
Apo form of protein shown in red and calcium-loaded form in blue.
Table 2.
Summary of melting temperatures of secretagogin and other calcium binding proteins.
Table 3.
Various buffer conditions used to study stability of secretagogin.