CLICKMASSLINK

Probing the organization of proteins and of their interactions within a living system is essential to understanding the role of these proteins in vivo. In the case of many pathogens, such as N. meningitidis, virulence is primarily dependent on the expression of filamentous organelles called type IV pili. The basic component of these pili is a single protein known as the major pilin, organized in a helical structure generating a flexible and mechanically resistant fiber. It has been shown that different cell conditions lead to differently organized pili and modulation of virulence, but the precise organization is not yet known. Standard structural biology techniques, such as X-ray crystallography or NMR can only probe specifically purified and crystallizable protein complexes. Thus obtaining structural data for a protein complex in its functional state remains a challenge. Mass spectrometry in combination with chemical cross-linking had been proposed a promising candidate for such an approach: the cross-linking reagents could be conceived as molecular rulers, whereas mass spectrometry, with its unique sensitivity and sequencing ability, could be used as a detector. Up to now, this approach has suffered from a number of drawbacks, which have limited its development as a mainstream technique in structural biology. This limited development is probably due to the fact that most of the chemical cross-linkers available are ‘off the shelf’ reagents, and are not specifically designed for the task. We propose in this project the synthesis and use of a new generation of chemical cross-linkers specifically designed for mass spectrometry in which a fixed charged group can be added by click-chemistry after the in vivo cross-linking step. Thus the first aim of this project is the design and synthesis of trifunctional cross-linking reagents bearing two protein reactive groups and one azido function. A fixed charge group will be added through the reaction of the click cross-linker, leading to enhanced ionization of these peptides vs. the non modified ones, and, as shown by one of the partner of the project, to increased sequence coverage when combined with electron capture dissociation. The design and use of modular reagents, in a ‘click-chemistry’ approach, will facilitate the optimization of the different steps of the methodology, as the initial experimental step could be optimized once, and a range of various groups attached when required in the detection protocol could be tested. Such an approach could also be conceived from the start by the addition of different functional groups on a single initial sample, allowing multiplexing experiments on a single sample. A first application of this methodology will be the structural analysis of type IV pili virulence complex expressed by the bacterial pathogen Neisseria meningitidis. The application of our innovative cross-linkers to type IV pili of N. meningitidis will give us the unique opportunity to study the different levels of structural organization of this virulence factor and more particularly to understand the structural role of posttranslational modifications. Multidisciplinarity is at the heart of this project as it is entirely dependent on the close collaboration between scientists in the field of organic chemistry, analytical chemistry and microbiology to solve biological questions relevant to biology and medicine. Organic synthesis chemists will bring the knowledge and ability to build new, custom designed molecules. Specialists of high resolution mass spectrometry (and experts in combining fixed charge derivatization and efficient fragmentation techniques) will bring both the technique, but also the knowledge on its advantages and current limitations as applied to the use of cross-linking. A microbiologist specialized in the study of bacterial pathogens brings biological questions on the organisation and the structure of virulence factors in the context of human pathology.