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In a rational protein design approach, the His3 Zn(II)-binding site from the active center of human carbonic anhydrase II was transplanted on the beta-barrel of mammalian serum retinol-binding protein (RBP) in a solvent-accessible location on the protein's outer surface. Several mutants of RBP were generated and produced in Escherichia coli, and their Zn(II)-binding properties were investigated in equilibrium dialysis experiments. One mutant, RBP/H3(A), with His residues introduced at the positions 46, 54, and 56 in the polypeptide sequence was shown to bind Zn(II) specifically with a stoichiometry of 1 and a corresponding dissociation constant equal to 36 +/- 10 nM. Binding of Zn(II) had no influence on the binding of retinoic acid, a natural ligand of RBP. In guanidinium chloride-induced unfolding experiments the mutant was found to be significantly stabilized in the presence of small concentrations of ZnSO4. This effect could be quantitatively explained using thermodynamic theory. Furthermore, it was demonstrated that the protein-bound Zn(II) is accessible to iminodiacetic acid as an additional chelating ligand without competition for the metal ion. Thus it was possible to use the grafted metal-binding site for the efficient purification of the engineered, bifunctional RBP via immobilized metal affinity chromatography from the bacterial protein extract.

Although the His3 metal-binding site used here had been removed from the substrate pocket of an enzyme and exposed to solvent on a protein surface, it showed clear selectivity for Zn(II) compared to Cu(II) and Ni(II). Thus the properties of this structurally defined metal-binding site (which are not shared by isolated His residues or flexible oligo-His tags) can be preserved when it is added to proteins. An IMAC matrix with improved behaviour was designed, allowing highly selective purification of RBP/H3(A) and of His6-tagged RBP as well. Such rational design of supramolecular recognition may be generally useful in the fields of protein engineering and drug design.

Naturally occurring fructosamines are of high clinical significance due to their potential use in diabetes mellitus monitoring (quantification of fructosylated hemoglobin, HbA 1c ) or for the investigation of their reactivity in consecutive reactions and harmfulness towards the organism. Here we report the specific synthesis of the fructosylated dipeptide L-valyl-L-histidine (Fru-Val-His) and fructosylated L-valine (Fru-Val). Both are basic tools for the development and validation of enzymatic HbA 1c assays. The two fructosamine derivatives were synthesized via a protected glucosone intermediate which was coupled to the primary amine of Val or Val-His, performing a reductive amination reaction. Overall yields starting from fructose were 36% and 34% for Fru-Val and Fru-Val-His, respectively. Both compounds were achieved in purities > 90%. A HILIC-ESI-MS/MS method was developed for routine analysis of the synthesized fructosamines, including starting materials and intermediates. The presented method provides a well-defined and efficient synthesis protocol with purification steps and characterization of the desired products. The functionality of the fructosylated dipeptide has been thoroughly tested in an enzymatic HbA 1c assay, showing its concentration-dependent oxidative degradation by fructosyl-peptide oxidases (FPOX).

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