- Role: Professore Associato
- Assignments:Teaching activity: Metodologie Biochimiche e Biomolecolari (Laurea triennale in Biotecnologie)
- Office:U3 - V piano- 5065
- Telephone number:02- 64483363
- Reception Hours:lun dalle 16 alle 17
Research projects of the group
Amyloid protein aggregation
Many pathological states are caused by protein misfolding and aggregation into amyloid fibrils, a process that can be reproduced in vitro. In order to understand the molecular mechanisms of this process, it is essential to describe its early stages, by identifying amyloidogenic conformers and soluble oligomers. The high complexity and the dynamic nature of such systems represent a major challenge in this kind of investigation. Mass spectrometry (MS) has developed into one of the central tools of this field, thanks to the possibility to detect non-covalent interactions and to identify distinct components of highly heterogeneous systems. Complementary information is gained by conventional biophysical techniques, such as circular dichroism and fluorescence spectroscopy. We apply nano-electrospray ionization (nano-ESI) as a mild ionization technique to analyze amyloid proteins under conditions that trigger aggregation, in order to identify intermediate states of the process and to analyze the influence of environmental variables. The proteins currently under investigation in our lab include human alpha-synuclein (collaboration with Prof. Giuseppe Legname, SISSA, Triest, Italy), beta-2-microglobulin (collaboration with Prof. Martino Bolognesi, University of Milan, Milan, Italy), and Ataxin-3 (collaboration with Prof. Paolo Tortora, University of Milano-Bicocca, Milan, Italy).
Intrinsically disordered proteins
Intrinsically disordered proteins (IDPs) lack ordered three-dimensional structure and undergo folding upon binding to specific interactors. They play key regulatory roles inside the cell and typically bind to multiple partners. In order to understand the mechanism of molecular recognition, it is essential to achieve structural characterization, not only of the ordered bound state, but also of the dynamic and elusive free state of these proteins. We focus on the cyclin-dependent protein kinase inhibitor Sic1 of the yeast Saccharomyces cerevisiae, which regulate the timing of entrance into the S phase controlling cell-cycle progression. Again, we use MS and other biophysical techniques to study the conformational properties of such a dynamic system, in the absence and in the presence of its interactors.
The mechanism of protein ionization by electrospray
The wide application of ESI-MS to proteomics and protein science contrasts with the relatively poor understanding of the process that leads to production of gas-phase protein ions during electrospray. We approach this issue, on one side, by dissecting the contributions of physico-chemical parameters like pH, surface tension and pI on the ionization behavior of reference peptides and proteins, in order to test alternative hypotheses on the ESI mechanism. On the other side, we make use of computational methods in order to simulate the desolvation and protonation processes and to model structure and ionization states of protein ions generated by electrospray.
PROTEINS BIOCHEMISTRY AND BIOPHYSICS: FUNCTION, INTERACTIONS, CONFORMATION
Dettagli Progetti di Ricerca
PROTEIN ENGINEERING AND INDUSTRIAL ENZYMOLOGY
Enzymes employed in biocatalysis, in particular lipases, and model proteins such as lactoglobulins are studied by a combined approach of mutagenesis (both directed evolution and random mutagenesis), heterologous expression, biochemical and biophysical characterization, in order to highlight the molecular bases of their stability, function and propensity to aggregate upon expression in bacterial cells. Cold adapted enzymes are used as models to understand the structural determinants of activity at low temperature. Moreover, novel biocatalysts are isolated from non commercial sources or produced by protein engineering.
Partecipants: Brocca Stefania , Lotti Marina
PROTEIN STRUCTURE, STABILITY AND AGGREGATION STUDIED BY FT-IR SPECTROSCOPY
Infrared absorption spectroscopy is used to obtain structural information on proteins in different environments, in aqueous and non aqueous solution, in solid form of pharmaceutical formulations, and within intact bacterial cells. In particular, FT-IR spectroscopy is employed to characterize the secondary structure of recombinant proteins , the stability of their different secondary elements during thermal unfolding, and the extent of glycosylation. Protein aggregation in form of inclusion bodies (IBs) in recombinant bacteria is also studied to monitor the kinetics of aggregation in vivo and to characterize the IB structural properties. Model peptides for amyloid proteins involved in neurodegenerative diseases are also investigated to identify intermediate species in the fibril formation process and to characterize at molecular level the fibril structural properties.
Partecipants: Doglia Silvia Maria
PROTEOMICS AND BIOPOLYMER MASS SPECTROMETRY
Mass spectrometry is employed on one side as an analytical tool for proteomics. The focus is on intracellular interactions and phosphorylation of the inhibitors of the cyclin-dependent protein kinases during the yeast cell cycle. On the other side, mass spectrometry is applied to the direct investigation of non-covalent interactions in intact protein structures for conformational studies and binding analysis. Finally, model proteins and peptides are employed for the investigation of the mechanism of electrospray ionization and for the study of non-covalent interactions in the gas phase.
Partecipants: Grandori Rita
STRUCTURE, PHYSIOLOGICAL ROLE AND PATHOGENIC POTENTIAL OF AMYLOIDOGENIC PROTEINS
Partecipants: Fusi Paola Alessandra , Regonesi Maria Elena , Tortora Paolo
Erika Ponzini – Ph. D. Student
room 4045, IV floor, building U3
tel: +39 02 6448 3458
The PhD School in Nanostructures & Nanotechnologies at the Department of
Materials Science of the University of Milano Bicocca: http://www.nano.unimib.it/