RICERCA ITALIANA     

Research program

Identification of folding and misfolding determinants by site-directed mutagenesis.

University Co-ordinator

Università degli Studi di NAPOLI "Federico II" - CHIMICA ORGANICA E BIOCHIMICA - ()

Research Unit Leader

Leila Birolo

Description

Within the framework of this research program we will exploit the techniques and know-how developed at our research group during the last decade, based on the application of advanced mass spectrometric methodologies to the conformational analysis on mutants of pathogenic protein beta2-microglobulin. Furthermore a central issue of our program will be the analysis of the interaction of beta2-microglobulin with collagen.
The main goal of this Unit will be to provide a structural support of alternative and complementary strategies for the conformational analysis of the beta2-microglobulin mutants prepared by Pavia Unit. These mutants will be analysed by other methods by the Units, in the perspective that this multi-Units integrated approach is nicely suited for challenging issues such as the analysis of transient intermediates in dynamic processes as protein conformational changes or misfolding as well as for the definition of the core of protein fibrils.
Transient intermediates, in fact, are molecular species termodinamically unstable and then difficult to analyse. To overcome these difficulties, new integrated strategies such as H/D exchange or limited proteolysis (LP) coupled to Mass Spectrometry (MS) have been developed [17, 21, 22]. The kinetics of the amide proton/deuterium exchange mainly depend on both solvent accessibility and stabilization of the associated protein backbone regions. Amide hydrogens provide specific targets along the entire polypeptidic chain to be exchanged with deuterium. However, the exchange rate at peptide amide linkages is strongly dependent on the localization of individual amino acids; amide protons occurring in structured portions of the protein and involved in stabilising hydrogen bonds show slow exchange kinetics, whereas those present in flexible regions display fast H/D exchange. Since MS analysis is able to measure the increase in mass associated with the exchange, the amount of deuterium incorporation and the kinetics of H/D exchange can be related to conformational changes of proteins in solution during the folding/unfolding process or under different experimental conditions such as protein-ligand complex formation (such as beta2-m – collagen complex formation).
Comparative H/D exchange measurements can then be used to monitor protein structural changes. Although hydrogen exchange has most often been measured by NMR, monitoring by MS has become increasingly common. In this respect, mass spectrometry has the unique advantage compared to NMR of being able to analyse the exchange behaviour of coexisting conformation in mixture.
Moreover, although never reaching the molecular resolution of NMR, HD/MS experiments can also provide information on individual regions of the polypeptide chain. Following H/D exchange, the labelled protein is submitted to proteolytic digestion with pepsin under acidic conditions that minimise proton exchange and the fragments generated are then analysed by liquid chromatography-mass spectrometric (LC-MS) methodologies. Peptides are identified by their unique mass values and by comparison with the fragments generated by the native protein. The increase in the molecular mass of the labelled fragments is directly linked to the number of incorporated deuterium atoms thus providing information on the solvent exposition of specific regions of the protein.
Conformational changes in proteins can also be investigated using limited proteolysis in a combined procedure where it is integrated with MS (LP/MS). The rationale behind this approach, is that the three-dimensional structure of proteins provides a stereochemical barriers to enzymatic attack, leaving only the exposed and flexible regions accessible to proteases. When these experiments are performed using a series of proteases with different specificities followed by mass spectrometric analysis of the fragments generated, the preferred cleavage sites will be strongly indicative of the exposed regions in the protein molecule. Conformational changes occurring in protein structure during the folding process can then be monitored by changes in the patterns of preferential cleavage sites, leading to the identification of the regions involved in the variations. Although this approach provide low-resolution data, it is amenable to the investigation of transient species or partly folded intermediates. This strategy can also be applied to the investigation of protein-ligand complexes. The main structural feature of protein-protein interaction, in fact, is that the regions involved are accessible to the solvent in the isolated molecules but they become protected following the formation of the complex. When limited proteolysis experiments are carried out on both the isolated proteins and the complex, differential peptide maps are obtained from which the definition of the interface zones can be extrapolated.

Within the framework of this research program we will systematically analyse beta2-mutants prepared at Pavia research Unit by H/D exchange experiments analysed by mass spectrometry, thus providing insights in the effect determined by single mutations on the overall flexibility of the protein. Pepsin digestion of the labelled mutants followed by MS analysis will give details of those protein regions that are solvent-exposed, or involved in conformational changes, or flexible enough to be therefore accessible to protein–protein interactions, thus probably seeding polimerization. This will therefore provide a “local” flexibility profile of the protein molecules.
This systematic approach will then be accompanied by limited proteolysis experiments on interesting mutants providing further details of the structural changes occurring upon mutation.
Moreover, a similar approach will be used in the investigation of the interaction of beta2- m (or its mutants), with collagen.
The results collected at our Research Unit will be complementing the structural studies carried out by NMR on the same mutants at the Research Unit of Udine, that will be continuously consulted to compare the results, and provide insights that might be instrumental also to understand at molecular level stability analysis on the mutants that will be carried out at the Research Unit of Firenze.