Ultrafast electron transfer in the complex between fluorescein and a cognate engineered lipocalin protein, a so-called anticalin.

Anticalins are a novel class of engineered ligand-binding proteins with tailored specificities derived from the lipocalin scaffold. The anticalin FluA complexes fluorescein as ligand with high affinity, and it effects almost complete quenching of its steady-state fluorescence. To study the underlying mechanism, we have applied femtosecond absorption spectroscopy, which revealed excited-state electron transfer within the FluA*Fl complex to be responsible for the strong fluorescence quenching. On the basis of a comparison of redox potentials, either tryptophan or tyrosine may serve as electron donor to the bound fluorescein group in its excited singlet state, thus forming the fluorescein trianion radical within 400 fs. The almost monoexponential rate points to a single, well-defined binding site, and its temperature independence suggests an (almost) activationless process. Applying conventional electron transfer theory to the ultrafast forward and slower back-rates, the resulting electronic interaction is rather large, with approximately 140 cm(-1) for tyrosine, which would be consistent with a coplanar arrangement of both aromatic moieties within van der Waals distance. The weak residual steady-state fluorescence originates from a small (approximately 10%) component with a time constant in the 40-60 ps range. These results demonstrate the power of time-resolved absorption spectroscopy as a diagnostic tool for the elucidation of a fluorescence quenching mechanism and the temporal profiles of the processes involved. The high structural and dynamic definition of the complexation site suggests the anticalin FluA to be a promising model in order to tailor and probe electronic interactions and energetics in proteins.

A novel type of receptor protein, based on the lipocalin scaffold, with specificity for digoxigenin.

We demonstrate that the bilin-binding protein, a member of the lipocalin family of proteins, can be structurally reshaped in order to specifically complex digoxigenin, a steroid ligand commonly used for the non-radioactive labelling of biomolecules. 16 amino acid residues, distributed across the four loops which form the binding site of the bilin-binding protein, were subjected to targeted random mutagenesis. From the resulting library the variant DigA16 was obtained by combined use of phage display and a filter-sandwich colony screening assay, followed by in vitro affinity maturation. DigA16 possesses strong binding activity and high specificity for the digoxigenin group, with a K(D) of 30.2(+/-3.6) nM. The derivative compound digitoxigenin is bound even more tightly, with a K(D) of 2.0(+/-0.52) nM, whereas the steroid glycoside ouabain is not recognized at all. Fusion proteins between DigA16 and alkaline phosphatase were constructed and shown to retain both the digoxigenin-binding function and enzymatic activity, irrespective of whether the enzyme was fused to the N or the C terminus of the bilin-binding protein variant. Our findings suggest that the lipocalin scaffold can be generally employed for the construction of specific receptor proteins, so-called "anticalins", which provide a promising alternative to recombinant antibody fragments.

Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold.

We demonstrate that the ligand pocket of a lipocalin from Pieris brassicae, the bilin-binding protein (BBP), can be reshaped by combinatorial protein design such that it recognizes fluorescein, an established immunological hapten. For this purpose 16 residues at the center of the binding site, which is formed by four loops on top of an eight-stranded beta-barrel, were subjected to random mutagenesis. Fluorescein-binding BBP variants were then selected from the mutant library by bacterial phage display. Three variants were identified that complex fluorescein with high affinity, exhibiting dissociation constants as low as 35.2 nM. Notably, one of these variants effects almost complete quenching of the ligand fluorescence, similarly as an anti-fluorescein antibody. Detailed ligand-binding studies and site-directed mutagenesis experiments indicated (i) that the molecular recognition of fluorescein is specific and (ii) that charged residues at the center of the pocket are responsible for tight complex formation. Sequence comparison of the BBP variants directed against fluorescein with the wild-type protein and with further variants that were selected against several other ligands revealed that all of the randomized amino acid positions are variable. Hence, a lipocalin can be used for generating molecular pockets with a diversity of shapes. We term this class of engineered proteins "anticalins." Their one-domain scaffold makes them a promising alternative to antibodies to create a stable receptor protein for a ligand of choice.

The three-dimensional structure of 6-phospho-beta-galactosidase from Lactococcus lactis.

BACKGROUND: The enzyme 6-phospho-beta-galactosidase hydrolyzes phospholactose, the product of a phosphor-enolpyruvate-dependent phosphotransferase system. It belongs to glycosidase family 1 and no structure has yet been published for a member of this family. RESULTS: The crystal structure of 6-phospho-beta-galactosidase was determined at 2.3 A resolution by multiple isomorphous replacement, using the wild-type enzyme and a designed cysteine mutant. A second crystal form, found with the mutant enzyme, was solved by molecular replacement, yielding the conformation of two chain loops that are invisible in the first crystal form. The active center, located through catalytic residues identified in previous studies, cannot be accessed by the substrate if the two loops are in their defined conformation. The enzyme contains a (beta alpha)8 barrel and the relationship of its chain fold to that of other glycosidases has been quantified. As a side issue, we observed that a cysteine point mutant designed for X-ray analysis crystallized mainly as a symmetric dimer around an intermolecular disulfide bridge formed by the newly introduced cysteine. CONCLUSIONS: The presented analysis provides a basis on which to model all other family 1 members and thereby will help in elucidating the catalytic mechanisms of these sequence-related enzymes. Moreover, this enzyme belongs to a superfamily of glycosidases sharing a (beta alpha)8 barrel with catalytic glutamates/aspartates at the ends of the fourth and the seventh strands of the beta barrel.