Probing factors affecting the gas phase stabilities of noncovalent complexes formed by peptides bound to the Grb2 SH2 domain protein

Abstract

Using collision-induced dissociation (CID) we have probed the gas phase dissociation energetics of noncovalent complexes formed between the SH2 domain of Grb2 and a range of high-affinity tyrosine-phosphorylated peptides. The chemical structures of the peptides were varied by modifying the N-terminus, which is in solution not involved in the binding pocket. CID experiments were performed both on the positively charged, 8-fold protonated, and negatively charged, 7-fold deprotonated, noncovalent complexes. For comparison, the solution phase binding of the Grb2 SH2 protein with these peptides was evaluated by surface plasmon resonance and electrospray mass spectrometry titration experiments indicating that the peptide modifications did not influence the solution phase binding. In contrast, we observed striking differences in the order of the dissociation energies for the four studied protein–peptides complexes, for both the positively and negatively charged ions, and additionally no clear correlation could be established between the order of the gas phase dissociation energies and the determined binding constants in solution. We argue that the observed differences can be rationalized by considering the energetics of the dissociation reactions, whereby especially the energy of the formed fragmentation products drives, in this case, the outcome of the CID reactions. Thus, the gas phase dissociation experiments presented here do not probe directly the strength of the noncovalent interactions between the protein and the peptide in the complex, but rather, the energetics of the fragmentation products.