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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

SEC-MALS results of HPC2 Cterm and SUPC2 Ccore in Ca2+-bound holo and Ca2+-free apo states.A, three different amounts of HPC2 Cterm protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-saturating buffer (20 mm CaCl2). The UV curves indicate the elution peaks of the protein samples analyzed, and the dotted lines indicate the calculated molar mass across the elution peaks. Orange, 1.16-mg injection; green, 155-μg injection; blue, 31.0-μg injection. B, three different amounts of HPC 2Cterm protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-free buffer (no added CaCl2, 1 mm EDTA). Orange, 2.36-mg injection; green, 650-μg injection; blue, 260-μg injection. C, three different amounts of SUPC2 Ccore protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-saturating buffer (20 mm CaCl2). The UV curves indicate the elution peaks of the protein samples analyzed, and the dotted lines indicate the calculated molar mass across the elution peaks. Orange, 1.35-mg injection; green, 150-μg injection; blue, 15.0-μg injection. D, three different amounts of SUPC2 Ccore protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-free buffer (no added CaCl2, 1 mm EDTA). Orange, 1.35-mg injection; green, 150-μg injection; blue, 75.2 μg injection.
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Figure 2: SEC-MALS results of HPC2 Cterm and SUPC2 Ccore in Ca2+-bound holo and Ca2+-free apo states.A, three different amounts of HPC2 Cterm protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-saturating buffer (20 mm CaCl2). The UV curves indicate the elution peaks of the protein samples analyzed, and the dotted lines indicate the calculated molar mass across the elution peaks. Orange, 1.16-mg injection; green, 155-μg injection; blue, 31.0-μg injection. B, three different amounts of HPC 2Cterm protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-free buffer (no added CaCl2, 1 mm EDTA). Orange, 2.36-mg injection; green, 650-μg injection; blue, 260-μg injection. C, three different amounts of SUPC2 Ccore protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-saturating buffer (20 mm CaCl2). The UV curves indicate the elution peaks of the protein samples analyzed, and the dotted lines indicate the calculated molar mass across the elution peaks. Orange, 1.35-mg injection; green, 150-μg injection; blue, 15.0-μg injection. D, three different amounts of SUPC2 Ccore protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-free buffer (no added CaCl2, 1 mm EDTA). Orange, 1.35-mg injection; green, 150-μg injection; blue, 75.2 μg injection.

Mentions: The addition of imidazole allows for measurement of the absolute molar mass of the HPC2 Cterm oligomer. Based on its molar mass, the oligomeric states of the HPC2 Cterm in solution were then determined under both apo (1 mm EDTA) and holo (20 mm CaCl2) conditions. Under each condition, different quantities of the HPC2 Cterm were analyzed to examine whether its oligomeric states are dependent on protein concentration. Under both conditions, similar ranges of molecular weight, determined by static light scattering, were observed for all three ranges of eluting concentrations (Fig. 2, A and B). The averaged values of molar mass were very similar for both apo and holo conditions, 96.5 kDa (apo) and 95.0 kDa (holo) (Table 1), respectively. With a monomeric mass of 32.2 kDa, the HPC2 Cterm forms a trimer independent of protein concentration over the tested range, with or without bound Ca2+. Based on parallel analyses of the SUPC2 Ccore, the protein appears to have a similar range of molar mass across three concentrations under both apo and holo conditions (Fig. 2, C and D). Because the monomer of the SUPC2 Ccore protein is predicted to have a mass of 21.3 kDa, the averaged molar mass for the two states obtained by static light scattering, 65.3 kDa (apo) and 67.0 kDa (holo), also suggests that the SUPC2 Ccore forms a trimer in solution, independent of its concentration in the examined range and the presence of Ca2+.


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)

SEC-MALS results of HPC2 Cterm and SUPC2 Ccore in Ca2+-bound holo and Ca2+-free apo states.A, three different amounts of HPC2 Cterm protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-saturating buffer (20 mm CaCl2). The UV curves indicate the elution peaks of the protein samples analyzed, and the dotted lines indicate the calculated molar mass across the elution peaks. Orange, 1.16-mg injection; green, 155-μg injection; blue, 31.0-μg injection. B, three different amounts of HPC 2Cterm protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-free buffer (no added CaCl2, 1 mm EDTA). Orange, 2.36-mg injection; green, 650-μg injection; blue, 260-μg injection. C, three different amounts of SUPC2 Ccore protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-saturating buffer (20 mm CaCl2). The UV curves indicate the elution peaks of the protein samples analyzed, and the dotted lines indicate the calculated molar mass across the elution peaks. Orange, 1.35-mg injection; green, 150-μg injection; blue, 15.0-μg injection. D, three different amounts of SUPC2 Ccore protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-free buffer (no added CaCl2, 1 mm EDTA). Orange, 1.35-mg injection; green, 150-μg injection; blue, 75.2 μg injection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: SEC-MALS results of HPC2 Cterm and SUPC2 Ccore in Ca2+-bound holo and Ca2+-free apo states.A, three different amounts of HPC2 Cterm protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-saturating buffer (20 mm CaCl2). The UV curves indicate the elution peaks of the protein samples analyzed, and the dotted lines indicate the calculated molar mass across the elution peaks. Orange, 1.16-mg injection; green, 155-μg injection; blue, 31.0-μg injection. B, three different amounts of HPC 2Cterm protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-free buffer (no added CaCl2, 1 mm EDTA). Orange, 2.36-mg injection; green, 650-μg injection; blue, 260-μg injection. C, three different amounts of SUPC2 Ccore protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-saturating buffer (20 mm CaCl2). The UV curves indicate the elution peaks of the protein samples analyzed, and the dotted lines indicate the calculated molar mass across the elution peaks. Orange, 1.35-mg injection; green, 150-μg injection; blue, 15.0-μg injection. D, three different amounts of SUPC2 Ccore protein were analyzed by Superdex 200 SEC-UV/LS/RI in Ca2+-free buffer (no added CaCl2, 1 mm EDTA). Orange, 1.35-mg injection; green, 150-μg injection; blue, 75.2 μg injection.
Mentions: The addition of imidazole allows for measurement of the absolute molar mass of the HPC2 Cterm oligomer. Based on its molar mass, the oligomeric states of the HPC2 Cterm in solution were then determined under both apo (1 mm EDTA) and holo (20 mm CaCl2) conditions. Under each condition, different quantities of the HPC2 Cterm were analyzed to examine whether its oligomeric states are dependent on protein concentration. Under both conditions, similar ranges of molecular weight, determined by static light scattering, were observed for all three ranges of eluting concentrations (Fig. 2, A and B). The averaged values of molar mass were very similar for both apo and holo conditions, 96.5 kDa (apo) and 95.0 kDa (holo) (Table 1), respectively. With a monomeric mass of 32.2 kDa, the HPC2 Cterm forms a trimer independent of protein concentration over the tested range, with or without bound Ca2+. Based on parallel analyses of the SUPC2 Ccore, the protein appears to have a similar range of molar mass across three concentrations under both apo and holo conditions (Fig. 2, C and D). Because the monomer of the SUPC2 Ccore protein is predicted to have a mass of 21.3 kDa, the averaged molar mass for the two states obtained by static light scattering, 65.3 kDa (apo) and 67.0 kDa (holo), also suggests that the SUPC2 Ccore forms a trimer in solution, independent of its concentration in the examined range and the presence of Ca2+.

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