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PubMed · 14572645

A binding event converted into a folding event.

F M Martín-Sierra·A M Candel·S Casares·V V Filimonov·J C Martínez·F Conejero-Lara
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Abstract

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.

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Amino Acid SequenceBinding SitesCalorimetry, Differential ScanningCircular DichroismHydrogen-Ion ConcentrationLigandsModels, MolecularMolecular Sequence DataProlineProtein BindingProtein DenaturationProtein FoldingProto-Oncogene Proteins c-ablRecombinant Fusion ProteinsSpectrinSpectrometry, FluorescenceStructural Homology, ProteinTemperatureThermodynamicssrc Homology DomainsComparative StudyJournal ArticleResearch Support, Non-U.S. Gov't

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F M Martín-Sierra, A M Candel, S Casares, V V Filimonov, J C Martínez, F Conejero-Lara. 2003-10-23. A binding event converted into a folding event.. https://doi.org/10.1016/s0014-5793(03)01038-x

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