[Design and structural and thermodynamic studies of a chimeric protein derived from spectrin SH3-domain].

A number of chimerical constructions based on the spectrin SH3 domain were designed for structural and thermodynamic studies of protein folding and protein-ligand interactions. SH3 domains were found in many regulatory proteins and operate through weak interactions with proline-rich fragments of the partners. The recombinant protein studied in this work (WT-CIIA) was constructed by linking the peptide PPPVPPYSAG to the domain C-terminal trough a long 12-residue linker with the aim to achieve stable ligand binding in orientation II, which until now has not been considered as typical for spectrin domain. A comparison of fluorescence spectra of the chimerical protein and the parent domain suggests that the ligand sticks to the conservative binding site. The analysis of the urea-induced unfolding curves revealed, however, that the protein-ligand contact is not stable enough and as a result the chimerical protein structure unfolds in two steps. In order to clarify the structural aspects of the protein-ligand interaction, WT-CIIA was crystallized and a set of the X-ray diffraction data at 1.75 angstroms resolution was acquired. Preliminary analysis of the diffraction data indicated that the crystals belong to the space group P32, with unit-cell parameters a = b = 3639, c = = 112.17 angstroms, alpha = beta = 90.0, gamma = 120.0.