Binding Site Mapping in Biomolecular Complexes

Biological processes are controlled, on a molecular level, by specific interactions of biomolecules. For the basic understanding of biochemical processes, it is essential to not only determine the structure of a biomolecule by itself, but to also characterize their interactions in biomolecular complexes. Likewise, the identification of binding sites in biomolecular complexes is highly important in the search for pharmacological agents that interact with a target molecule instead of a natural binding partner. There are several experimental approaches for the study of interactions between biomolecules, among them X-ray structure analysis and nuclear magnetic resonance spectroscopy. A relatively new and evolving method for the characterization of biomolecules is modern mass spectrometry. The aim of this project is the realization of two complementary strategies for the identification of binding sites in biomolecular complexes using mass spectrometry methods. Both strategies are based on radical chemistry immediately after complex desolvation. In the first approach, electron transfer induces radical reactions in the intermolecular binding interface region of the complex, resulting in local backbone cleavages of the biomolecule. The analysis of the biomolecule fragments by ultra-high resolution mass spectrometry then identifies the amino acid residues or nucleotides in the binding region, and a direct imprint of the interaction site is obtained. In contrast, the second approach relies on radical chemistry on the exposed complex surface, resulting in stable oxidative modifications for proteins and backbone cleavage for nucleic acids. The mass spectral analysis of the oxidation and cleavage products then identifies the amino acid residues and nucleotides on the complex surface, thereby providing a complementary "negative" imprint of the interaction site of the complex partners. The mass spectrometry approach in both methods has the special advantage that it can use unmodified, non-labeled compounds. Moreover, the use of mass spectrometry makes both methods considerably faster and more sensitive than conventional techniques, and in principle offers the potential for automation. The model systems for the development and validation of both methods include protein- as well as nucleic acid-complexes of major biological significance and topicality.