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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

NMR spectra of SUPC2 Ccore under holo and apo conditions. Comparison of 1H-15N HSQC NMR spectra of 13C15N SUPC2 Ccore in the Ca2+-saturating condition (red contours) and in the Ca2+-free condition (green contours) in pH 7.4, 2 mm Tris-d11, 150 mm KCl buffer with 1 mm TCEP at 25 °C.
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Figure 4: NMR spectra of SUPC2 Ccore under holo and apo conditions. Comparison of 1H-15N HSQC NMR spectra of 13C15N SUPC2 Ccore in the Ca2+-saturating condition (red contours) and in the Ca2+-free condition (green contours) in pH 7.4, 2 mm Tris-d11, 150 mm KCl buffer with 1 mm TCEP at 25 °C.

Mentions: To map the Ca2+-binding elements in the SUPC2 Ccore structure, two-dimensional HSQC and a series of three-dimensional NMR experiments were collected with this protein under both holo and apo conditions. In the spectra collected under holo conditions, the majority of the peaks were well resolved and dispersed with different chemical shifts, clearly indicating that the SUPC2 Ccore sample is well folded under saturating Ca2+ conditions (Fig. 4A). The majority of the well resolved peaks were assigned based on three-dimensional 13C-15N-1H experiment spectra in SUPC2 Ccore. Nevertheless, there were an insufficient number of peaks in the three-dimensional NMR spectra to account for all the residues in the SUPC2 Ccore protein. For instance, there are only 145 peaks in the HNCO spectrum, whereas there are 192 residues in the SUPC2 Ccore construct. After residue assignment, we found that most of the assigned peaks belong to residues in the EF-hand and L2 regions of the SUPC2 Ccore protein. The peaks that belong to residues in the downstream coiled-coil region are not present in these spectra, possibly due to the line broadening from a combination of slow rotation rate and conformational exchange. In the 1H-15N HSQC spectra recorded under holo conditions, the peaks corresponding to the residues in the EF-hand region appear to have much narrower line widths than what would be expected for a trimeric protein of 64 kDa. Instead, the line widths of these peaks are comparable with that for the monomeric SUPC2 C-EF protein, suggesting that each EF-hand domain moves on a faster time scale compared with the rest of the protein.


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)

NMR spectra of SUPC2 Ccore under holo and apo conditions. Comparison of 1H-15N HSQC NMR spectra of 13C15N SUPC2 Ccore in the Ca2+-saturating condition (red contours) and in the Ca2+-free condition (green contours) in pH 7.4, 2 mm Tris-d11, 150 mm KCl buffer with 1 mm TCEP at 25 °C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400361&req=5

Figure 4: NMR spectra of SUPC2 Ccore under holo and apo conditions. Comparison of 1H-15N HSQC NMR spectra of 13C15N SUPC2 Ccore in the Ca2+-saturating condition (red contours) and in the Ca2+-free condition (green contours) in pH 7.4, 2 mm Tris-d11, 150 mm KCl buffer with 1 mm TCEP at 25 °C.
Mentions: To map the Ca2+-binding elements in the SUPC2 Ccore structure, two-dimensional HSQC and a series of three-dimensional NMR experiments were collected with this protein under both holo and apo conditions. In the spectra collected under holo conditions, the majority of the peaks were well resolved and dispersed with different chemical shifts, clearly indicating that the SUPC2 Ccore sample is well folded under saturating Ca2+ conditions (Fig. 4A). The majority of the well resolved peaks were assigned based on three-dimensional 13C-15N-1H experiment spectra in SUPC2 Ccore. Nevertheless, there were an insufficient number of peaks in the three-dimensional NMR spectra to account for all the residues in the SUPC2 Ccore protein. For instance, there are only 145 peaks in the HNCO spectrum, whereas there are 192 residues in the SUPC2 Ccore construct. After residue assignment, we found that most of the assigned peaks belong to residues in the EF-hand and L2 regions of the SUPC2 Ccore protein. The peaks that belong to residues in the downstream coiled-coil region are not present in these spectra, possibly due to the line broadening from a combination of slow rotation rate and conformational exchange. In the 1H-15N HSQC spectra recorded under holo conditions, the peaks corresponding to the residues in the EF-hand region appear to have much narrower line widths than what would be expected for a trimeric protein of 64 kDa. Instead, the line widths of these peaks are comparable with that for the monomeric SUPC2 C-EF protein, suggesting that each EF-hand domain moves on a faster time scale compared with the rest of the protein.

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