Investigation on protein aggregation in natural and artificial models.

Abstract

In this Thesis, two proteins, RNase A and MNEI have been proposed as models for aggregation studies. Although different in structure and in function, they share common hallmarks that make them useful models to study the influenace of chemical,genetic and physical agents on protein stability and aggregation in vitro. RNase A is presented as a model to study the metalation of proteins. The interaction between RNase A and Pt drugs improves protein oligomerization. Pt oligomers are endowed with peculiar characteristics such us SDS resistance and prolonged stability in solution. These results might suggest alternative aggregation mechanisms different from the well-known domain swapping. Pt-drugs selected also affect the biological action of RNase A evaluated both as enzymatic activity in a standard assay and also as cytotoxicity on tumoral cells.This suggest that the Pt-drugs deeply influence the structure and the function pf the protein as explained by solving the crystallographic structure of the cisplatinated monomer and of the carboplatinated monomer adducts. Further investigation on dimers may allow obtain structural information on Pt-dependent dimerization. RNase A is also presented as a helpful model to design new mutant with different structural features. The structural characterization of the mutant RNase A-Onc showed that shortening of the C-terminal hinge loop does not affect the global folding but can improve the aggregation propensity that, in this protein occurs spontaneously. Very likely, in this case 3D-DS is involved. Biophysical studies on dimers obtained in native conditions allow to characterise them and to compare their structures with the well-known swapped dimers. In this thesis, the artificial model MNEI was also presented as a suitable system. It represents a very fascinating model to study the influence of several physicochemical factors. It has been proven that pH, temperature and ionic strength deeply influence MNEI solubility and the aggregation towards amyloid-like patterns. The aggregation mechanism is worth of investigation whit the aim to obtain ordered assemblies under controlled conditions or avoid their formation and preserve the native structural and functional elements.