17th International Mass Spectrometry Conference :: Prague, 2006
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|Presentation date:||Mon, Aug 28, 2006|
|Presentation time:||09:50 – 11:20|
Montserrat Morell Fernandez1, Marc Martinell2, Francesc Xavier Aviles1, Ernest Giralt21 Institut de Biotecnologia i Biomedicina, Departament de Bioquimica, Cerdanyola del Valles, Spain
Correspondence address: Montserrat Morell Fernandez, Institut de Biotecnologia i Biomedicina, Campus de la Universitat Autonoma de Barcelona, Cerdanyola del Valles, 08193 Spain.
Keywords: Combinatorial Library; Digests, Enzymatic or Proteolytic; High Throughput; Interactions, Protein/Peptide.
Novel aspect: Use of exoproteolysis and mass spectrometry analysis for ligand screening.
One powerful strategy for deducing protein function is to identify the interacting partners of proteins. Several methods to identify protein-protein interactions at large scale have been developed, like two-hybrid screenings, protein chips, TAP approach or pull down assays. In previous works, we developed a method to screen ligands using a combination of exoproteolysis and mass spectrometry analysis.1 In this work, we have used the same approach to screen a set of peptide ligands against the tetramerization domain (TD) of the p53 protein. Human protein p53 is a sequence-specific homotetrameric transcription factor, which acts as a natural tumour suppressor controlling cell cycle progression, apoptosis and DNA repair.2 It is involved in approximately 50% of all human cancers. As many transcription factors, it contains several domains. One of them is the tetramerization domain that permits the oligomerization of the protein and its binding to DNA. Since tetramerization is essential for p53 function, small molecules that bind to this domain and affect its stability could potentially be used to modulate the antitumour activity of the protein. From a tetraguanidinium compound that binds to p53 tetramerization domain (TD),3 a peptide that also interacts with this domain has been designed. This interaction is based on electrostatic interactions between peptide arginines and the carboxylate-rich protein domain surface. After that, a peptide library has been made varying functional groups and chain lengths to improve the binding of these peptide ligands. Applying exoproteolysis and mass spectrometry analysis, relative binding values have been determined for the different peptides tested against p53 TD. The obtained affinity order is quite similar to the order obtained by fluorescence measurements demonstrating the applicability of this method for the characterization of protein-ligand interaction.
1. Villanueva, et al., J. Mol. Biol. 330, 1039 (2003).
2. Yako, et al., Trends Biochem. Sci. 27, 612 (2002).
3. Salvatella, et al., Angew. Chem. Int. Ed. Engl. 43, 196 (2004).