Premium
Stability and peptide binding affinity of an SH3 domain from the Caenorhabditis elegans signaling protein Sem‐5
Author(s) -
Lim Wendell A.,
Fox Robert O.,
Richards Frederic M.
Publication year - 1994
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560030812
Subject(s) - sh3 domain , caenorhabditis elegans , guanidine , denaturation (fissile materials) , biophysics , chemistry , guanine nucleotide exchange factor , crystallography , peptide , signal transduction , biochemistry , biology , proto oncogene tyrosine protein kinase src , nuclear chemistry , gene
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.