A binding event converted into a folding event

• Published in: FEBS letters Vol. 553; no. 3; pp. 328 - 332
• Main Authors: Martı́n-Sierra, F.M, Candel, A.M, Casares, S, Filimonov, V.V, Martı́nez, J.C, Conejero-Lara, F
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 23.10.2003
• Subjects: Amino Acid Sequence; Binding Sites; Calorimetry, Differential Scanning; CD, circular dichroism; Circular Dichroism; Differential scanning calorimetry; DSC, differential scanning calorimetry; Hydrogen-Ion Concentration; Ligands; Models, Molecular; Molecular Sequence Data; p41, decapeptide of sequence APSYSPPPPP; Proline - genetics; Proline - metabolism; Proline-rich peptide; Protein Binding; Protein Denaturation; Protein Folding; Protein stability; Protein–ligand binding; Proto-Oncogene Proteins c-abl - chemistry; Proto-Oncogene Proteins c-abl - genetics; Proto-Oncogene Proteins c-abl - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; S19P20s, circular permutant of the α-spectrin SH3 domain cut between positions S19 and P20 with its N- and C-ends linked; SH3, Src-homology region 3; SPCp41, chimeric S19P20s elongated from its C-terminus with a three-residue link of sequence DGN plus the p41 sequence; Spectrin - chemistry; Spectrin - genetics; Spectrin - metabolism; Spectrometry, Fluorescence; src Homology Domains; Src-homology region 3 domain; Structural Homology, Protein; Temperature; Thermodynamics; Differential scanning calorimetry; S19P20s, circular permutant of the α-spectrin SH3 domain cut between positions S19 and P20 with its N- and C-ends linked; DSC, differential scanning calorimetry; Protein folding; Src-homology region 3 domain; SPCp41, chimeric S19P20s elongated from its C-terminus with a three-residue link of sequence DGN plus the p41 sequence; Protein stability; SH3, Src-homology region 3; p41, decapeptide of sequence APSYSPPPPP; Proline-rich peptide; Protein–ligand binding; CD, circular dichroism
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(03)01038-X

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.