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Structural Aspects of Ligand Recognition by Engineered Lipocalins

Structural Aspects of Ligand Recognition by Engineered Lipocalins [Pg.199]

The 3-D structures of the fluorescein- and digoxigenin-binding BBP variants have recently been analyzed by X-ray crystallography and compared with the original bi-hn-binding protein. The crystal structures were determined in different space groups and, for one variant, in both the presence and absence of the hapten, thus [Pg.199]

In the case of the fluorescein-binding variant FluA, crystals were obtained in the presence of the hgand at pH 8.1 with two FluA fluorescein complexes in the asymmetric unit, which were refined to a resolution of 2.0 A [52]. The two molecules were highly similar in structure, with a root mean square difference (rmsd) of 0.33 A for 173 mutually superimposed positions. The overall topology of the //-barrel with the a-hehx attached to it, both of which are characteristic features of the lipocahn architecture (see Section 8.2), was found to be conserved (Fig. 8.4). Both disulfide bonds of the BBP scaffold, one between Cys and fJys and one between Cys and Cys, were also clearly visible. Upon superposition with the BBP crystal structure (molecule A from the Protein Data Base entry IBBP [32]), an rmsd of 1.2 A was calculated for 159 superimposed positions. [Pg.200]

Fluorescein is bound at the bottom of the cleft that harbors bihverdin IX, in the wild-type BBP structure (Fig. 8.2). Its xanthenolone moiety is located close to the center of the j8-barrel, while the carboxyphenyl group is oriented towards the entrance of the pocket. The para ring position (with respect to the central carbon atom of the triphenylmethane dye), which carried the linker group during the selection experiments for this anticalin [40], is accessible from the solvent via a narrow charmel. An area of 454 A , corresponding to 91% of the solvent-accessible surface of fluorescein, became buried in the complex. [Pg.201]

Approximately 50% of the buried area from the side of the protein is contributed by 6 of the 16 residues that were mutated in the generation of FluA from BBP. The remaining buried surface belongs to 10 residues that were not mutated. Unexpectedly, when compared with the complexation of biliverdin by BBP, fluorescein was found to be inserted even more deeply into the hydrophobic core of the -barrel. In the central region of the protein, where no mutations had been introduced, the necessary space was created by the movement of loop 3 and by rearrangement of several side chains. [Pg.201]




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Engineering aspects

Ligand aspects

Ligand recognition

Ligand structures

Ligands ligand structure

Lipocalin

Lipocalins

Structural engineering

Structural engineers

Structural recognition

Structures of ligands

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