This study described the interaction of the HPK 1 analog peptides with the SH3 domain of cortactin.Results generally revealed that the replacement of Pro residues into the P2 sequence influences in different ways the conformational properties of peptides in both aqueous and organic solvents.But, neither the presence of N-alkyl moieties nor the presence of the proline homologous Pip increase the peptide binding affinity to cortactin-SH3 domain.