A binding event converted into a folding event

FEBS Letters

Conejero-Lara, F¹  Martı́nez, J.C¹  Filimonov, V.V¹  Candel, A.M¹  Casares, S¹  Martı́n-Sierra, F.M¹ 

[1]Departamento de Quı́mica Fı́sica e Instituto de Biotecnologı́a, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain

关键词: Src-homology region 3 domain;    Proline-rich peptide;    Protein–ligand binding;    Protein folding;    Protein stability;    Differential scanning calorimetry;    SH3;    Src-homology region 3;    p41;    decapeptide of sequence APSYSPPPPP;    S19P20s;    circular permutant of the α-spectrin SH3 domain cut between positions S19 and P20 with its N- and C-ends linked;    SPCp41;    chimeric S19P20s elongated from its C-terminus with a three-residue link of sequence DGN plus the p41 sequence;    DSC;    differential scanning calorimetry;    CD;    circular dichroism;   

DOI  :  10.1016/S0014-5793(03)01038-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We have designed a chimeric protein by connecting a circular permutant of the α-spectrin SH3 domain to the proline-rich decapeptide APSYSPPPPP with a three-residue link. Our aim was to obtain a single-chain protein with a tertiary fold that would mimic the binding between SH3 domains and proline-rich peptides. A comparison of the circular-dichroism and fluorescence spectra of the purified chimera and the SH3 circular permutant showed that the proline-rich sequence occupies the putative SH3 binding site in a similar conformation and with comparable interactions to those found in complexes between SH3 and proline-rich peptides. Differential scanning calorimetry indicated that the interactions in the binding motif interface are highly cooperative with the rest of the structure and thus the protein unfolds in a two-state process. The chimera is more stable than the circular permutant SH3 by 6–8 kJ mol⁻¹ at 25°C and the difference in their unfolding enthalpy is approximately 32 kJ mol⁻¹, which coincides with the values found for the binding of proline-rich peptides to SH3 domains. This type of chimeric protein may be useful in designing SH3 peptide ligands with improved affinity and specificity.

【 授权许可】

Unknown