We have designed a chimeric protein by connecting a circular permutant of the K K-spectrin SH3 domain to the prolinerich 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 £uores-cence spectra of the puri¢ed 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. Di¡erential 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 31 at 25 ‡C and the di¡erence in their unfolding enthalpy is approximately 32 kJ mol 31 , 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 a⁄nity and speci¢city.

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