A binding event converted into a folding event.
We have designed a chimeric protein by connecting a circular permutant of the alpha-spectrin SH3 domain to the proline-rich decapeptide APSYSPPPPP with a three-residue link. Our aim was to obtain a single-chain protein with a tertiary fold that would mimic the binding between SH3 domains and proline-rich peptides. A comparison of the circular-dichroism and fluorescence spectra of the purified chimera and the SH3 circular permutant showed that the proline-rich sequence occupies the putative SH3 binding site in a similar conformation and with comparable interactions to those found in complexes between SH3 and proline-rich peptides. Differential scanning calorimetry indicated that the interactions in the binding motif interface are highly cooperative with the rest of the structure and thus the protein unfolds in a two-state process. The chimera is more stable than the circular permutant SH3 by 6-8 kJ mol(-1) at 25 degrees C and the difference in their unfolding enthalpy is approximately 32 kJ mol(-1), which coincides with the values found for the binding of proline-rich peptides to SH3 domains. This type of chimeric protein may be useful in designing SH3 peptide ligands with improved affinity and specificity.
Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain.
F M
FM
Martín-Sierra
A M
AM
Candel
S
S
Casares
V V
VV
Filimonov
J C
JC
Martínez
F
F
Conejero-Lara
D000595:Amino Acid Sequence; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011392:Proline / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D016315:Proto-Oncogene Proteins c-abl / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D040681:Structural Homology, Protein; D013696:Temperature; D013816:Thermodynamics; D018909:src Homology Domains
D008024:Ligands; D011993:Recombinant Fusion Proteins; D013049:Spectrin; D011392:Proline; D016315:Proto-Oncogene Proteins c-abl