Protein Science, Vol 3, Issue 8 1261-1266, Copyright © 1994 by Cold Spring Harbor Laboratory Press

ARTICLE

Stability and peptide binding affinity of an SH3 domain from the Caenorhabditis elegans signaling protein Sem-5

W. A. LIM, R. O. FOX and F. M. RICHARDS
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511

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{deg}C. Interestingly, the protein is not maximally stable at neutral pH, but reaches a maximum at around pH 4.7 (T(m) {complex} 80{deg}C). Increasing ionic strength also stabilizes the protein, suggesting that 1 or more carboxylate 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{deg}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) {complex} 30 {mu}M), 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.
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