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Oligomerization of the polycystin-2 C-terminal tail and effects on its Ca2+-binding properties.

Yang Y, Keeler C, Kuo IY, Lolis EJ, Ehrlich BE, Hodsdon ME - J. Biol. Chem. (2015)

Bottom Line: Consequently, trimerization does not further improve the affinity of Ca(2+) binding in the SUPC2 Ccore relative to the isolated EF-hand domain.Our study provides a structural basis for understanding the Ca(2+)-dependent regulation of the PC2 channel by its cytosolic C-terminal domain.The improved methodology also serves as a good strategy to characterize other Ca(2+)-binding proteins.

View Article: PubMed Central - PubMed

Affiliation: From the Departments of Laboratory Medicine, Pharmacology, and yifei.yang@yale.edu.

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Related in: MedlinePlus

ITC measurement of PC2 C-terminal constructs and Ca2+ binding interaction.A, raw heat measurement of 1.96 mm CaCl2 titrated into 100 μm HPC2 Cterm protein in pH 7.4, 25 mm Tris, 150 mm KCl, 20 mm imidazole buffer at 25 °C, using 32 injections of 1.49 μl/injection. B, raw heat measurement of 1.96 mm CaCl2 titrated into 98.5 μm HPC2 Cterm protein and 115 μm EDTA in pH 7.4, 25 mm Tris, 150 mm KCl, 20 mm imidazole buffer at 25 °C, using 32 injections of 1.49 μl/injection. C, simultaneously fitted, baseline-corrected isotherms of CaCl2 into HPC2 Cterm only (blue trace) and protein with EDTA (red trace) ITC experiment. D, raw heat measurement of 1.00 mm CaCl2 titrated to 15.2 μm SUPC2 C-EF protein in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 36 injections of 8 μl/injection. E, raw heat measurement of 1.00 mm CaCl2 titrated into 13.3 μm SUPC2 C-EF protein and 88 μm 5,5′-dimethyl-BAPTA in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 36 injections of 8 μl/injection. F, simultaneously fitted, baseline-corrected isotherms of SUPC2 C-EF only (blue trace) and SUPC2 C-EF with 5,5′-dimethyl-BAPTA (red trace) ITC experiment. G, raw heat measurement of 2.44 mm CaCl2 titrated into 88 μm SUPC2 Ccore protein in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 32 injections of 1.49 μl/injection. H, raw heat measurement of 2.44 mm CaCl2 titrated into 86 μm SUPC2 Ccore protein and 255 μm EDTA in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 32 injections of 1.49 μl/injection. I, simultaneously fitted, baseline-corrected isotherms of SUPC2 Ccore only (blue trace) and SUPC2 Ccore with EDTA (red trace) ITC experiment.
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Figure 3: ITC measurement of PC2 C-terminal constructs and Ca2+ binding interaction.A, raw heat measurement of 1.96 mm CaCl2 titrated into 100 μm HPC2 Cterm protein in pH 7.4, 25 mm Tris, 150 mm KCl, 20 mm imidazole buffer at 25 °C, using 32 injections of 1.49 μl/injection. B, raw heat measurement of 1.96 mm CaCl2 titrated into 98.5 μm HPC2 Cterm protein and 115 μm EDTA in pH 7.4, 25 mm Tris, 150 mm KCl, 20 mm imidazole buffer at 25 °C, using 32 injections of 1.49 μl/injection. C, simultaneously fitted, baseline-corrected isotherms of CaCl2 into HPC2 Cterm only (blue trace) and protein with EDTA (red trace) ITC experiment. D, raw heat measurement of 1.00 mm CaCl2 titrated to 15.2 μm SUPC2 C-EF protein in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 36 injections of 8 μl/injection. E, raw heat measurement of 1.00 mm CaCl2 titrated into 13.3 μm SUPC2 C-EF protein and 88 μm 5,5′-dimethyl-BAPTA in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 36 injections of 8 μl/injection. F, simultaneously fitted, baseline-corrected isotherms of SUPC2 C-EF only (blue trace) and SUPC2 C-EF with 5,5′-dimethyl-BAPTA (red trace) ITC experiment. G, raw heat measurement of 2.44 mm CaCl2 titrated into 88 μm SUPC2 Ccore protein in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 32 injections of 1.49 μl/injection. H, raw heat measurement of 2.44 mm CaCl2 titrated into 86 μm SUPC2 Ccore protein and 255 μm EDTA in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 32 injections of 1.49 μl/injection. I, simultaneously fitted, baseline-corrected isotherms of SUPC2 Ccore only (blue trace) and SUPC2 Ccore with EDTA (red trace) ITC experiment.

Mentions: To determine whether there are additional Ca2+-binding sites outside the EF-hand region, we needed to be certain that both the binding stoichiometry and the binding affinity of the HPC Cterm were measured with a high degree of accuracy. Because Ca2+ is necessary for the expression and purification of the protein, it is inherently difficult to maintain a Ca2+-free protein sample without the use of Ca2+ chelators. If the KD for the Ca2+-binding affinity of the protein lies in a low micromolar range, even a micromolar level of residual Ca2+ in the sample will render a significant proportion of protein unavailable for ITC characterization. To account for the residual Ca2+ bound to the protein, two ITC experiments were set up to pair each “Ca2+-HPC2Cterm” experiment (Fig. 3A) with a matching “Ca2+-HPC2Cterm + chelator” experiment (Fig. 3B). The thermodynamic parameters of the binding interaction were determined by fitting both binding isotherms simultaneously to an identical binding site model (Fig. 3C). The presence of the Ca2+ chelator enabled us for the first time to definitively determine the binding stoichiometry, N, by accounting for the effects of residual Ca2+. The best fit values for binding stoichiometry are very close to 1 (Table 2), indicating that there is only one Ca2+-binding site, presumed to be the same Ca2+-binding site previously identified in the HPC2 C-EF. The best fit value for the binding affinity KD is 22 μm. Compared with the known HPC2 C-EF binding affinity (Kd ∼461 μm) (17), the Ca2+-binding affinity is significantly enhanced (20-fold increase) in the longer and trimeric HPC2 Cterm construct.


Oligomerization of the polycystin-2 C-terminal tail and effects on its Ca2+-binding properties.

Yang Y, Keeler C, Kuo IY, Lolis EJ, Ehrlich BE, Hodsdon ME - J. Biol. Chem. (2015)

ITC measurement of PC2 C-terminal constructs and Ca2+ binding interaction.A, raw heat measurement of 1.96 mm CaCl2 titrated into 100 μm HPC2 Cterm protein in pH 7.4, 25 mm Tris, 150 mm KCl, 20 mm imidazole buffer at 25 °C, using 32 injections of 1.49 μl/injection. B, raw heat measurement of 1.96 mm CaCl2 titrated into 98.5 μm HPC2 Cterm protein and 115 μm EDTA in pH 7.4, 25 mm Tris, 150 mm KCl, 20 mm imidazole buffer at 25 °C, using 32 injections of 1.49 μl/injection. C, simultaneously fitted, baseline-corrected isotherms of CaCl2 into HPC2 Cterm only (blue trace) and protein with EDTA (red trace) ITC experiment. D, raw heat measurement of 1.00 mm CaCl2 titrated to 15.2 μm SUPC2 C-EF protein in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 36 injections of 8 μl/injection. E, raw heat measurement of 1.00 mm CaCl2 titrated into 13.3 μm SUPC2 C-EF protein and 88 μm 5,5′-dimethyl-BAPTA in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 36 injections of 8 μl/injection. F, simultaneously fitted, baseline-corrected isotherms of SUPC2 C-EF only (blue trace) and SUPC2 C-EF with 5,5′-dimethyl-BAPTA (red trace) ITC experiment. G, raw heat measurement of 2.44 mm CaCl2 titrated into 88 μm SUPC2 Ccore protein in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 32 injections of 1.49 μl/injection. H, raw heat measurement of 2.44 mm CaCl2 titrated into 86 μm SUPC2 Ccore protein and 255 μm EDTA in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 32 injections of 1.49 μl/injection. I, simultaneously fitted, baseline-corrected isotherms of SUPC2 Ccore only (blue trace) and SUPC2 Ccore with EDTA (red trace) ITC experiment.
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Figure 3: ITC measurement of PC2 C-terminal constructs and Ca2+ binding interaction.A, raw heat measurement of 1.96 mm CaCl2 titrated into 100 μm HPC2 Cterm protein in pH 7.4, 25 mm Tris, 150 mm KCl, 20 mm imidazole buffer at 25 °C, using 32 injections of 1.49 μl/injection. B, raw heat measurement of 1.96 mm CaCl2 titrated into 98.5 μm HPC2 Cterm protein and 115 μm EDTA in pH 7.4, 25 mm Tris, 150 mm KCl, 20 mm imidazole buffer at 25 °C, using 32 injections of 1.49 μl/injection. C, simultaneously fitted, baseline-corrected isotherms of CaCl2 into HPC2 Cterm only (blue trace) and protein with EDTA (red trace) ITC experiment. D, raw heat measurement of 1.00 mm CaCl2 titrated to 15.2 μm SUPC2 C-EF protein in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 36 injections of 8 μl/injection. E, raw heat measurement of 1.00 mm CaCl2 titrated into 13.3 μm SUPC2 C-EF protein and 88 μm 5,5′-dimethyl-BAPTA in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 36 injections of 8 μl/injection. F, simultaneously fitted, baseline-corrected isotherms of SUPC2 C-EF only (blue trace) and SUPC2 C-EF with 5,5′-dimethyl-BAPTA (red trace) ITC experiment. G, raw heat measurement of 2.44 mm CaCl2 titrated into 88 μm SUPC2 Ccore protein in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 32 injections of 1.49 μl/injection. H, raw heat measurement of 2.44 mm CaCl2 titrated into 86 μm SUPC2 Ccore protein and 255 μm EDTA in pH 7.4, 25 mm Tris, 150 mm KCl, 1 mm TCEP buffer at 25 °C, using 32 injections of 1.49 μl/injection. I, simultaneously fitted, baseline-corrected isotherms of SUPC2 Ccore only (blue trace) and SUPC2 Ccore with EDTA (red trace) ITC experiment.
Mentions: To determine whether there are additional Ca2+-binding sites outside the EF-hand region, we needed to be certain that both the binding stoichiometry and the binding affinity of the HPC Cterm were measured with a high degree of accuracy. Because Ca2+ is necessary for the expression and purification of the protein, it is inherently difficult to maintain a Ca2+-free protein sample without the use of Ca2+ chelators. If the KD for the Ca2+-binding affinity of the protein lies in a low micromolar range, even a micromolar level of residual Ca2+ in the sample will render a significant proportion of protein unavailable for ITC characterization. To account for the residual Ca2+ bound to the protein, two ITC experiments were set up to pair each “Ca2+-HPC2Cterm” experiment (Fig. 3A) with a matching “Ca2+-HPC2Cterm + chelator” experiment (Fig. 3B). The thermodynamic parameters of the binding interaction were determined by fitting both binding isotherms simultaneously to an identical binding site model (Fig. 3C). The presence of the Ca2+ chelator enabled us for the first time to definitively determine the binding stoichiometry, N, by accounting for the effects of residual Ca2+. The best fit values for binding stoichiometry are very close to 1 (Table 2), indicating that there is only one Ca2+-binding site, presumed to be the same Ca2+-binding site previously identified in the HPC2 C-EF. The best fit value for the binding affinity KD is 22 μm. Compared with the known HPC2 C-EF binding affinity (Kd ∼461 μm) (17), the Ca2+-binding affinity is significantly enhanced (20-fold increase) in the longer and trimeric HPC2 Cterm construct.

Bottom Line: Consequently, trimerization does not further improve the affinity of Ca(2+) binding in the SUPC2 Ccore relative to the isolated EF-hand domain.Our study provides a structural basis for understanding the Ca(2+)-dependent regulation of the PC2 channel by its cytosolic C-terminal domain.The improved methodology also serves as a good strategy to characterize other Ca(2+)-binding proteins.

View Article: PubMed Central - PubMed

Affiliation: From the Departments of Laboratory Medicine, Pharmacology, and yifei.yang@yale.edu.

Show MeSH
Related in: MedlinePlus