Anticalins

The main problems that require more research and optimization of immunosensor design are nonspecific adsorption of coexisting substances, further involvement of genetic engineering in production of novel antibodies, and search for the same purpose of smaller proteins with antibody properties (e.g., anticalins). 

Fig. 8.3 Gen eration of anticalins by randomization and selection. (Top) Randomized amino acid positions (corresponding side chains are shown in light blue) in the BBP, depicted...

The generation of BBP variants with novel binding specificities for fluorescein or digoxigenin, respectively, has demonstrated for the first time that a lipocalin can be tailored to recognize non-natural ligands. In order to illustrate the anti-body-like binding function of the engineered lipocalins, this new class of proteins was termed anticalins [40]. 

Fig. 8.4 Crystal structure of the anticalin FluA with the bound fluorescein (green). positions of the 16 randomized amino acids are shown as gray spheres. Trp is depicted with its side chain in magenta.

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. 

The crystal structure of the FluA fluorescein complex also provides a structural explanation for the strong quenching effect of this particular anticalin that was mentioned before. Tight coplanar packing of the indole ring of I rp against the xanthenolone system of the bound fluorescein was observed (Fig. 8.4). Consequently, this aromatic residue is the likely candidate for the highly efficient electron-transfer process and is optimally positioned in this respect. 

In the case of the anticalin DigA16, crystal structures were solved not only for the bound digoxigenin but also for the complex with the related steroid digitoxigenin and for the uncomplexed apo-protein [56]. The crystals, which were grown at pH 7.6-8.0 and whose structures were refined to resolutions between 1.8 and... 

Fig. 8.5 Molecular recognition of haptens by engineered lipocalins versus antibodies. (Top) Crystal structure of the anticalin DigAl 6 with the bound digoxigenin (yellow). The arrow points to the hydroxyl substituent of steroid ring A, which had served for covalent attachment - via a flexible spacer - to a solid support during the selection process for this BBP variant. The Cq positions of the 16 initially ran-...

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