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A binding event converted into a folding event
Author(s) -
Martı́n-Sierra F.M,
Candel A.M,
Casares S,
Filimonov V.V,
Martı́nez J.C,
Conejero-Lara F
Publication year - 2003
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(03)01038-x
Subject(s) - sh3 domain , circular dichroism , chemistry , peptide , protein folding , crystallography , proline , binding site , polyproline helix , binding domain , biophysics , stereochemistry , biochemistry , proto oncogene tyrosine protein kinase src , biology , amino acid , signal transduction
We have designed a chimeric protein by connecting a circular permutant of the α‐spectrin SH3 domain to the proline‐rich decapeptide APSYS 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.