Protein stabilization studied by mass spectrometry

This proposal describes new aims within the frame of the project "Mass-spectrometry for protein folding studies", which is currently funded by the Charlotte-Bühler Habilitationsstipendium H147-CHE. The research lines presented here generate from new results recently obtained in our group investigating protein-glycerol interactions by mass-spectrometry (MS). These results show that electrospray mass spectrometry (ESI-MS), with a turbo- ionspray sample interface, allows detection of protein ions from glycerol-containing aqueous solutions, which are not amenable to measurements by regular or nanoelectrospray interfaces. The results of our previous experiments can be summarized in the following three points. 1) Monitoring stabilization of lysozyme and cytochrome c by glycerol against different denaturing agents. 2) Detection of conformational changes accompaining lysozyme stabilization under native conditions. 3) Identification of partially folded conformations of the employed proteins. These results open the way to numerous experiments aimed to further investigation of protein stabilization by polyols. This proposal expands the original project, focusing on new applications of ESI-MS to the study of stabilization of native and partially folded conformations. Three new aims are described here. 1) Assessment of glycerol-induced changes on different proteins of known structures, in order to describe the relationship between glycerol effect and compactness and/or flexibility of the native structure. 2) Comparative analysis of protein stabilization by different polyols, for a quantitative comparison of different stabilizing agents. 3) Characterization of partially folded states of reference proteins, in order to develop a new method for fast identification of molten- globule-like conformations allowing for simultaneous discrimination from the native and denatured states. These studies should contribute to our understanding of the mechanism of protein stabilization and protein folding in solution. Conformational studies by MS could provide an effective new method for quick evaluation of compactness and quality of side-chain packing in globular structures.

Mass spectrometry (MS) allows detection of ions of an analyte of interest, after transfer to the gas phase. MS is a fast developing technique in protein science. Its applications go from protein identification to direct detection of intact supramolecular complexes. This project focused on the analysis of protein non-covalent interactions by MS, which is enabled by "soft" ionization methods like electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). The experiments were performed by a nano-ESI-MS equipment, which allows for particularly mild desolvation conditions. Non-covalent interactions mediate the biological function of proteins, i.e. the acquisition of a specific three-dimensional structure (folding), the interactions with cofactors, substrates, inhibitors and other macromolecules, and catalysis. We were interested, in particular, in the study of folding and binding reactions. The relevance to analyze these transitions by MS is based on a major advantage that is peculiar to this technique, namely the direct visualization of all the molecular species present in heterogeneous sample. Indeed, MS does not average over the molecular population, but rather provides distinct information on each component deriving from conformational or mass heterogeneity. This unique feature allows to monitor changes in the individual species involved in binding equilibria and to obtain combined information about folding and binding. We studied conformational and oligomeric properties of the arginine repressor (ArgR) from Escherichia coli. The results include detection of the intact hexamer, identification of the trimer as the intermediate of the assembly process and evidence of specific interactions between hexamers. These results acquire particular relevance considering that crystallographic data depict a poorly developed interface between trimers in the absence of L- arginine, which involves only hydrophobic interactions and no salt bridges or hydrogen bonds. The survival of such a complex in vacuo could be mediated by van der Waals interactions and by hydrogen bonding through trapped solvent molecules. Formation of ArgR dodecamers could be relevant for ArgR function. Finally, we investigated the linked equilibria of folding, subunit association, and ligand (FMN) binding in the multimeric flavodoxin-like protein WrbA from E. coli, a protein involved in the cellular defense against oxidative stress. Thermal unfolding of the apo- and holo-protein monitored by infrared spectroscopy indicated a dramatic effect of the non-covalently-bound flavin cofactor in protein stabilization. Analysis by nano-ESI-MS showed that the addition of the cofactor to the apo-protein shifts the oligomerization equilibrium towards the tetramer. Furthermore, it could be seen that monomers and dimers are selectively depleted at increasing temperature. Thus, it is possible to suggest a mechanism for the effect of the cofactor based on stabilization of the tetrameric form and enhanced thermoresistance of the latter compared to dimer and monomer.