Protein folding and binding studied by mass spectrometry

This research proposal focuses on applications of nano-electrospray ionization mass spectrometry to the study of protein non-covalent interactions, i.e. folding and binding. In particular, the unique advantages offered by this technique for the characterization of structurally heterogeneous samples will be exploited for the analysis of binding and folding equilibria by monitoring different conformational or oligomeric states. An exciting perspective for this kind of studies is application of protein mass spectrometry under mild conditions to quantitative analysis of dynamic equilibria. To this purpose, simple and well-characterized systems, like cytochrome c for folding and myoglobin for protein-cofactor binding, will be used to compare results obtained by mass spectrometry and other traditional methods. The analysis would then be extended to novel systems of biological relevance, such as interactions of apo- myoglobin with heme analogs; folding, oligomerization and flavin binding of a multimeric flavodoxin-like protein; folding, oligomerization and arginine binding of the hexameric arginine repressor; and folding of lipases and lipase mutants. Another relevant aspect of analyzing protein folding by mass spectrometry is the possibility to detect and characterize folding intermediates, thanks to the exceptional sensitivity of the method detecting poorly populated conformational states. Interesting results recently obtained on the cytochrome c molten globule motivate further application of this kind of studies to myoglobin, another protein following a three-state folding mechanism. The research plan can be summarized in the following four aims: 1) quantitative analysis of protein folding transitions, 2) characterization of folding intermediates, 3) quantitative analysis of binding to prosthetic groups, 4) studies on subunit interactions in oligomeric proteins.