Results And Discussion

Description of the Geometry of Natural EF-hand sites

To seek a common parameter set that can be used to identify the natural calcium binding sites, an ideal pentagonal bipyramidal geometry with bond length of Ca-O 2.4 A is used. As listed in Table 1, different types of oxygen ligands of natural calcium binding sites in calmodulin, parvalbumin and calbindinD9k have very similar Ca-O distances with an average value of 2.4 ± 0.4 A (Table 1). The bond lengths Ca-O therefore are constrained within a relatively narrow range from 1.5-3.5 (2.5 ± 1.0) A for design purposes. On the other hand, the angles of O-Ca-O differ largely in the natural calcium-binding sites (Table

2). In reference to the Oe atom of carboxylate bidentate Glu, the O-Ca-O angle for two ligand oxygen atoms on the same plane from the sidechain of Asp or Asn at sequence position 5 and from mainchain of carbonyl group at sequence position 7 vary from 71o to 130" (Table 2). The oxygen atom off the plane from the sidechain ligand (Asp or Asn) at sequence position 1 has O-Ca-O angles between 84o and 132o. To accommodate such great variations in O-Ca-O angle, a relatively open value with a range of 120" is used for the construction of potential sites. The O-Ca-O angles in an ideal pentagonal bipyramidal geometry for all the uni-dentate ligands on the plane and off the plane are centered at 72, 144 and 90 o, respectively. These values are close to the average values of the O-Ca-O angles in natural calcium binding proteins (Table 2). Due to the constraints for the two oxygen atoms within the same residue of Glu, the O-Ca-O angles for all bidentate ligand Glu in all three proteins are well defined with an average of 53 ± 3o. Besides the bidentate Glu and the water ligand, rotamer libraries of Asp, Asn and mainchain for 20 amino acids are used for the construction of calcium sites.

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