Nucleic acid hybridisation-directed control of peptide conformation as tool in the study of biological signal transduction

The majority of molecular interactions in signalling pathways is governed by Src homology 2 domains (SH2), non-catalytic protein modules which bind to phosphotyrosine loops within receptor tyrosine ki-nases (RTK). For example, the binding of the Grb2 SH2 domain to RTKs is required for the activation of the Ras-protein, a central switch in the signal transduction cascade. In the setting of this modular mode of protein-protein interaction, the protein framework serves to scaffold the peptide segments, thus, re-stricting conformational freedom. For a putative peptide ligand this scaffolding can be mimicked by pep-tide cyclisation or by grafting the peptide to loop-forming templates. In this research project, a novel class of switchable loop mimetics will be developed with the aim to inhibit or induce biological signal transduction pathways. To this end the interaction of phosphotyrosine containing sequences with SH2 domain proteins such as Grb 2 will be in the focus. It is the objective to develop peptide conjugates that are inactive in one conformational state but become bioactive upon an inducible conformational reor-ganisation. We propose to exert this conformational control by nucleic acid hybridisation. A peptide of interest will be equipped with peptide nucleic acid (PNA) sequences, which will flank both termini. For example, the resulting PNA-peptide chimera are designed to form a triple-helical stem-loop structure upon addition of an oligonucleotide. The stem will be provided by PNA units whereas the peptide part will span a bridging loop. This feature could prove particularly useful in real-time single-cell measure-ments for the study of signal transduction events in situ. As an example, "activated" phosphotyrosyl-PNA-peptide chimera may selectively sequester SH2-possessing molecules. "Activatable" PNA-peptide chimera will be identified by combinatorial chemistry, which will also provide a powerful methodology to identify novel recognition motifs.