Abstract
We have determined the thermodynamic stability and peptide binding affinity of the carboxy‐terminal Src homology 3 (SH3) domain from the Caenorhabditis elegans signal‐transduction protein Sem‐5. Despite its small size (62 residues) and lack of disulfide bonds, this domain is highly stable to thermal denaturation — at pH 7.3, the protein has a T~m~ of 73.1 °C. Interestingly, the protein is not maximally stable at neutral pH, but reaches a maximum at around pH 4.7 (T~m~ ≅ 80 °C). Increasing ionic strength also stabilizes the protein, suggesting that 1 or more car‐boxylate ions are involved in a destabilizing electrostatic interaction. By guanidine hydrochloride denaturation, the protein is calculated to have a free energy of unfolding of 4.1 kcal/mol at 25 °C. We have also characterized binding of the domain to 2 different length proline‐rich peptides from the guanine nucleotide exchange factor, Sos, one of Sem‐5′s likely physiological ligands in cytoplasmic signal transduction. Upon binding, these peptides cause about a 2‐fold increase in fluorescence intensity. Both bind with only modest affinities (K~d~ ≅ 30 μM), lower than some previous estimates for SH3 domains. By fluorescence, the domain also appears to associate with the homopolymer poly‐L‐proline in a similar fashion.