Stability and peptide binding affinity of an SH3 domain from the Caenorhabditis elegans signaling protein Sem-5.
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 Tm of 73.1 degrees C. Interestingly, the protein is not maximally stable at neutral pH, but reaches a maximum at around pH 4.7 (Tm approximately equal to 80 degrees 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 degrees 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 (Kd approximately equal to 30 microM), 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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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Fox
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Richards
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000737:chemistry*; D029742:Caenorhabditis elegans Proteins; D004220:Disulfides / Q000378:metabolism*; D004355:Drug Stability; D004563:Electrochemistry; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D015801:Helminth Proteins / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D015398:Signal Transduction; D013816:Thermodynamics
D029742:Caenorhabditis elegans Proteins; D004220:Disulfides; D006146:Guanidines; D015801:Helminth Proteins; D010455:Peptides; C085751:Sem-5 protein, C elegans; D019791:Guanidine