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* The value before minimization.

* The value before minimization.

As shown in Table 3, the relocated natural calcium binding sites have small U(p) values (<25). They all have the smallest U(p) values among all the constructed sites for each protein. Therefore, the U(p) number can be used to rank the constructed potential sites according to their deviations from the target site. Our parameters used to describe the geometry of calcium binding sites allow us to accurately identify natural calcium binding sites.

To evaluate the differences between the natural sites in calmodulin, parvalbumin and calbindinD9K with the defined parameters of the ideal pentagonal bipyramidal geometry, the coordinates of the natural classic EF-hand sites from these three proteins are submitted into the same minimization processes. The U(p) of all the natural sites with a classic EF-hand motif are from 5-12 (Table 3). The differences of the pseudo-energy values between the natural sites and relocated sites of calmodulin site 4 and parvalbumin site 2 are noticeably greater than those of the remaining sites we examined. For example, the relocated Site 4 of calmodulin has a U(p) value of 23.08, which is a significant difference from its natural site (5.44). As shown in Fig.5, quite a few other non-natural sites have U(p) values lower than that for this related Site 4 of calmodulin. In addition, this relocated site has the largest deviation for the O-Ca length and O-Ca-O angle from that of the natural sites (Table 1 and 2). This indicates that although the parameters used are opened enough to relocate the natural ligand type, they are not enough to obtain the best configuration set. When the bond length is enlarged from 1.5-3.5 to 1 .0-4.0 to research the site 4 of calmodulin, the newly relocated site 4 has the pseudo-energy of 5.26 after minimization, which almost equals that of the natural site 4. The O-Ca bond length and O-Ca-O angle are very similar to those of the ideal pentagonal bipyramidal and natural calmodulin site 4 (Table 1, 2 and 4). This newly obtained configuration of Site 4 was rejected at the Site-search step using length range of 1.5-3.5 because one of the O-Ca lengths of this relocated site (N129 OS-Ca) before minimization is only 1.248 Á (Table 4). It seems that the change from one configuration to another results in relatively large changes in length and angle, limited by the size of the rotamer library. Narrow parameters may reject some configurations that can be adjusted in the Refinement program.

Site Number

Figure 5. The deviation of the designed sites from the ideal pentagonal bipyramidal geometry (pseudo-energy U(p)) as a function the designed calcium binding sites. The natural calcium binding sites I-IV of calmodulin are shown as black dots.

Site Number

Figure 5. The deviation of the designed sites from the ideal pentagonal bipyramidal geometry (pseudo-energy U(p)) as a function the designed calcium binding sites. The natural calcium binding sites I-IV of calmodulin are shown as black dots.

The parameter set is defined by compromising between obtaining the required necessary combinations of configurations and the time used for the analysis. The parameter set with O-Ca length range of 1.5-3.5 A and 0-Ca-0 angle range of 120o is suitable for the relocation of EF-hand motifs with proper ligand types and geometry in a reasonable time. To identify some specific regions of the protein with large deviation from the target site, a more opened parameter set is preferred. On the other hand, if O-Ca length range and O-Ca-O angle are specifically defined according to those of the natural site and each ligand position is occupied by its corresponding residue, the natural site can be uniquely identified in a very short time.

Identification ofPseudo EF-hand site in calbindinD9K

We have also attempted to identify the pseudo-EF-hand site (Site 1) in calbindinD». Glu is again used as an anchor residue and the water ligand is constructed by leaving one position above the plane unoccupied. Since the remaining four positions in Site 1 are from the mainchain carbonyl groups, only the coordinates of the mainchain oxygen atoms are used for the construction of the pseudo EF-hand site. With the same parameter set, only one site is constructed and all the ligand types and residue numbers for the constructed site are identical to that of the natural calcium-binding site 1. As shown in Fig. 4, the geometric parameters for both angles and both lengths for the reconstructed pseudo-EF-hand site are very similar to that of natural calcium binding site 1. Although the U(p) number for the constructed site is about 40 and larger than that for all the reconstructed natural classic EF-hand sites in calmodulin, parvalbumin and site II of calbindinD9k, these relatively large values are generated by the relative weight factors oj we used for the shift of backbone atoms compared with that for the sidechain configurations in the calculation of U(p) with equation 1.

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