Table I.
Estimated secondary structure composition of RCK domains
| CD spectroscopy | X-ray diffraction | |||||
| BKCa RCK1, 0 Ca2+ | BKCa RCK1, 35 µM Ca2+ | BKCa RCK1-D362/367A | BKCa RCK1 | MthK RCK | MthK RCK Ca2+ bound | |
| Amino acids | 322IIE…HDP667, 346 aa | 343KHI…IEY570, 577SRI…YCK613, 265 aa | 116RHV…ISA336, 221 aa | |||
| Reference | This study | Yuan et al., 2010 | Ye et al., 2006 | Jiang et al., 2002 | ||
| α helix (%) | 28.1 ± 0.751 | 19.3 ± 0.557 | 24.8 ± 0.0882 | 39.6 (30) | 40.9 | 38.2 |
| nα helix | 10.3 ± 0.415 | 7.81 ± 0.288 | 9.26 ± 0.0302 | 14 | 12 | 13 |
| β strand (%) | 23.2 ± 0.318 | 30.2 ± 0.731 | 25.4 ± 0.208 | 18.5 (14) | 22.3 | 22.3 |
| nβ strand | 15.4 ± 0.181 | 18.3 ± 0.582 | 14.6 ± 0.396 | 12 | 12 | 12 |
| Turn + unordered (%) | 48.9 ± 0.503 | 46.8 ± 0.689 | 48.7 ± 0.306 | 41.9 (56) | 36.8 | 39.5 |
| NRMSD | 0.0630 ± 0.00900 | 0.0827 ± 0.0123 | 0.115 ± 0.00762 | |||
| Fold | α/β | α/β | α/β | α/β | α/β | α/β |
Secondary structure composition of the BKCa RCK1 and MthK RCK domains. Deconvolution of far-UV CD spectra performed on the purified WT-RCK1 in the presence and absence of Ca2+ and D362/367A-RCK1 into percent secondary structural contributions. The WT-RCK1 domain in 0.00058 µM Ca2+ has higher α-helical content than D362/367A-RCK1. The addition of Ca2+ to WT-RCK1 changes the secondary structure fractions of the RCK1 domain, decreasing the α-helix content and increasing the β-strand content (see Fig. 6). Secondary structure fractions of the crystallized BKCa RCK1 domain (Protein Data Bank accession no. 3MT5 estimated from the resolved regions 343KHI…IEY570 and 577SRI…YCK613) and the MthK RCK domain in the presence (Protein Data Bank accession no. 1LNQ) or absence (Protein Data Bank accession no. 2FY8) of Ca2+ are provided for reference, obtained from the DSSP reference set. Note that the purified RCK1 domain in this study is composed of 346 amino acids (322IIE…HDP667, which amounts to an 81–amino acid difference between the RCK1 domain in this study and the BKCa RCK1 x-ray structure, and a 126–amino acid difference with respect to the two MthK RCK domains). This region includes the flanking S6-RCK1 linker and part of the RCK1-RCK2 linker, which were not included or resolved in the BKCa crystal structure. The structure fractions reported in parentheses were estimated by assuming the unresolved RCK1 flaking linker regions possessed no additional α or β structures. Errors represent standard errors of the mean (n = 3). NRMSD, normalized root mean-square deviation.