Design and structural thermodynamic studies of the chimeric protein derived from spectrin SH3 domain

Autor: Azat Gabdulkhakov, Liubov V. Gushchina, Vladimir V. Filimonov
Rok vydání: 2009
Předmět:
Zdroj: Molecular Biology. 43:444-452
ISSN: 1608-3245
0026-8933
DOI: 10.1134/s0026893309030121
Popis: A number of the chimeric constructs with spectrin SH3 domain were designed for structural and thermodynamic studies of protein self-assembly and protein-ligand interactions. SH3 domains, components of many regulatory proteins, operate through weak interactions with proline-rich regions of polypeptide chains. The recombinant construct (WT-CIIA) studied in this work was constructed by linking the peptide ligand PPPVPPYSAG to the domain C-terminus via a long 12-residue linker to increase the affinity of this ligand for the spectrin domain, thereby ensuring a stable positioning of the polyproline helix to the conserved ligand-binding site in orientation II, which is regarded as untypical of the interaction between this domain and oligopeptides. A comparison of fluorescence spectra of the initial domain and the recombinant protein WT-CIIA suggests that the ligand sticks to the conservative binding site. However, analysis of the equilibrium urea-induced unfolding has demonstrated that this is an unstable contact, which leads to a two-stage unfolding of the chimeric protein. The protein WT-CIIA was crystallized; a set of X-ray diffraction data with a resolution of 1.75 A was recorded from individual crystals. A preliminary analysis of these diffraction data has demonstrated that the crystals belong to space group P32 with the following unit cell parameters: a = b = 36.39, c = 112.17 A, a = β = 90.0, and γ = 120.0.
Databáze: OpenAIRE
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