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A de novo designed calcium-binding site into the framework of a non-calcium-binding protein, domain 1 of CD2, is introduced. The feasibility of designing a single calcium-binding site into a host protein, taking into account only local properties of a calcium-binding site obtained by a survey of natural calcium-binding proteins and chelators has been demonstrated.

Author(s): Loren L. Looger; Mary A. Dwyer; James J. Smith; Homme W. Hellinga (corresponding author) The formation of complexes between proteins and ligands is fundamental to biological processes at the [...]

Research suggests that the open and closed conformations of maltose-binding protein facilitate variable copper-copper distance formation. A mutation in the hinge region of the protein leads to the formation of a binuclear copper center, which simulates oxy-hemocyanin upon reacting with hydrogen peroxide.

Factors that influence the energy landscape of the Blue Copper metalloprotein compound were investigated. The Blue Copper CysHis2Met primary coordination sphere was placed in different orientations within the hydrophobic core of thioredoxin and an iterative design procedure that can be utilized to define dominant competing states was presented. Results showed that destabilization of competing reactions is a critical aspect of molecular recognition in metalloproteins.

A family of seven single, mononuclear Cys2His2 zinc sites was constructed in various areas in the hydrophobic core of Escherichia coli thioredoxin using a semi-automated, rational design approach. Test results show that the technique allows researchers to examine attributes that affect metal binding and protein stabilization. In addition, the capability of a protein fold to accomodate metal-mediated cross links was confirmed.

A metal-based redox center in the Escherichia coli protein, thioredoxin, was characterized using a design based on a mononuclear iron-sulfur center. The design was characterized by the conversion of a disulfide into a metal-based redox center generated by the Dezymer automatic design algorithm. The iron complex was determined by direct titration with iron through the reconstitution of iron-sulfur proteins. A geometrical accident of history led to the conversion of a pre-existing redox-active disulfide bridge into a tetrahedral Cys4 mononuclear iron center.

Protein engineering techniques such as the transmission of conformational changes in a protein can be applied in integrating optical signal transduction functions directly into proteins. This is done by converting a ligand binding event into a change in fluorescence using an allosteric coupling mechanism. Fluorescent allosteric signal transduction sites can then be designed into the Glucose/Galactose Binding Protein of Escherichia coli, which can form the basis for the development of a new class of fluorescent glucose sensors.

EPD-Fe-mediated protein cleavage technique is reliable for the conformational analysis of staphylococcal nuclease. The cleavage technique reveals the presence of solvent-accessible residues on the protein surface at a distance of 15 angstrom units from the cysteine binding sites. The cleavage technique yields data on the integrity of the tertiary protein fold, denatured protein states and secondary structural elements and indicates the existence of an equilibrium folding intermediate when the denaturant levels are low.

The structural variation of the thioredoxin protein in a disulfide active site of Escherichia coli due to oxdiation/reduction reaction is detected by differential scanning calorimetry. The active site contains a dithiol along with the disulfide. Isosteric double mutation process and thermal denaturation process are explained. The structural changes retard calculation of thermodynamic parameters.

pH ranging from 6 to 8 influences the thermodynamics of the reversible thermal exhibition of Escherichia coli thioredoxin in the oxidized state. The ph for different concentrations is obtained by the use of differential scanning calorimetry. Thermal denaturation reveals that temperature dependence is inversely proportional to the concentration.