Development of mass spectrometry for the dynamic study of non-covalent complexes in biology and in chemistry

The achievement of stability constants in addition to structural information is essential for a complete characterization of non-covalent complexes. The further characterization of these buildings is necessary both to optimize their synthesis (Supramolecular Chemistry) and to understand their function (structure-function relationships in biology). In this context, our project is mainly oriented towards the development of mass spectrometry to characterize mechanisms of formation of chemical and biological supramolecular systems and to determine their stability constants. To be able to determine the stability constants of these buildings by mass spectrometry remains a challenge, even stronger than the conventional analytical techniques (NMR, spectrophotometry, surface plasmon resonance) sometimes have limits. Today supramolecular mass spectrometry is primarily a tool for determining the nature and stoichiometry of assembly of the partners.It is rarely used for following the complexes formation (kinetics study) and for determining their stability constants. So our goal is through the study of different systems to determine the conditions under which mass spectrometry can obtain such information by demonstrating in particular that the mass spectrum gives the qualitative and quantitative image of the species present in solution (in including comparison of the results obtained with other techniques). The deep characterization of these complexes involves also defining their interaction zones. For this, we identify by mass spectrometry the amino acids that are in proximity to each other in each partner of the complex. These approaches consist to achieve the peptide mapping of all the partners after the chemical cross-linking of the complex in its native conformation.

Stability constants determination of protein-protein complexes

This study is a fundamental study to evaluate and to use the ability of mass spectrometry to determine the stability constants of protein-protein complexes, using different protein/protein complexes on which the determination of stability constants by traditional approaches was impossible (too fast kinetics of complexation). This work has been performed in collaboration with Prof. Rita Bernhardt (Universität des Saarlandes). Stability constants have been obtained on different AdX/AdR complexes (including AdX mutations); combined with protein-protein docking, the mass spectrometry data haven given informations on how the different partners interact with each other.

Study of the formation of high molecular weight protein complexes

In the field of respiratory biology, the goal of this work is to determine the influence of the biotope physicochemical parameters (oxygen, sulfide, pressure, temperature) on the formation of these protein complexes composed by the assembly of several protein units with molecular weights up beyond 1 Million Daltons (collaboration with F. Zal and A. Andersen, Station Biologique de Roscoff).The results obtained on the stoichiometry of the complexes should better understand the adaptive capacity of organisms to extreme environments. In this context, complexes denaturation in solution has been followed by mass spectrometry.

From mass measurement to protein topology

Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this project, we combine chemical cross-linking with mass spectrometry (CX-MS) to determine the binding stoichiometry and map the protein-protein interaction network of protein complexes.

Chemical cross-linking is carried out in order to stabilize the complex prior to MS analyses. After cross-linking, a bottom up approach is performed and the intra- and intercross-linked peptides are identified using dedicated search engines.

The data generated by CX-MS will provide associations and restraints information that, in combination with complementary analytical methods, might help to build coherent structural models.

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