Titolo della tesi: Unveiling folding and fibrillar aggregation properties of KH domains: a comparative analysis
KH domains are one of the most important and well-represented RNA binding domains in proteins, both in prokaryotes and eukaryotes, involved in a variety of crucial biological functions ranging from cellular homeostasis to embryonic and adult development. In line with their pivotal physiological role, the dysregulation of KH domains binding capacity is reported to determine the onset of several pathologies. Despite the ability of proteins to fold properly, to misfold, or to aggregate is of paramount importance for their cellular functions, there are no studies about the folding mechanism of any of the known KH domains, neither in procaryotes nor in eukaryotes. The present Ph. D. thesis aims at filling this gap, providing the first analysis on the folding mechanism and aggregation/misfolding properties of the KH domains. For this purpose, the prokaryotic single KH domain-constituted Ribosome binding factor A from the opportunistic pathogen Pseudomonas aeruginosa (RbfA), and the eukaryotic KH domains KH0 and KH1 from the Fragile X Mental Retardation Protein (FMRP-KH0 and FMRP-KH1), were used as model systems. Thanks to a complementary and multidisciplinary approach spanning from molecular biology to biochemistry and biophysics techniques, the folding mechanism of the three proteins were explored, also highlighting the remarkable ability of RbfA and FMRP-KH0 to form fibrillar aggregates in mild conditions. Finally, pathological variants of the Fragile X Mental Retardation Protein affecting the FMRP-KH0 (R138Q) and FMRP-KH1(G266E) and which are known to cause Fragile X-like syndrome in patients, were analyzed, finding that these mutations alter the aggregation properties (R138Q) or native fold (G266E) of the relative wild type domains.