Joined Application Of Computational And Experimental Methods To The Structural And Dynamic Study Of Proteins

During the past three decades, Molecular Dynamics (MD) computer simulations have considerably contributed to our understanding at the atomic level of the properties of molecular systems. MD simulation results can sometimes be compared directly with experimental ones and thus, they become an extremely powerful tool not only to understand and interpret the experiments at the microscopic level, but also to study regions which are not accessible experimentally. In the present thesis computational and experimental methods we jointly applied to the study of three complex and biologically relevant model systems: the Human Topoisomerase IB (HTop1), an enzyme able to control DNA topological states; the Cu,Zn Superoxide dismutase from Haemophilus ducreyi (HdSOD), having the unique ability among SODs of binding an heme molecule; and the Human Prion Protein (HuPrP), an ubiquitary glycoprotein whose misfolding is known to cause several neurodegenerative diseases. MD simulations were performed, for the Htop1 system, to investigate the effects of the substitution of a polar residue with a smaller, non polar one in the hinge region on which the protein hinges to open and close around the incoming DNA double strand. Analyses proved to be able to disclose the structural an dynamic features underling the observed experimental behaviour of the wild type and mutated systems. The study of the effect of the binding of a CO molecule at the heme group at subunits interface of HdSOD required the construction of an ad hoc force field and the conformational sampling obtained was tested by checking its ability to reproduce the experimental data collected by means of XAFS. The MD simulations evidenced “long range effect” of the gaseous molecule binding, involving the active site. Finally, XAFS technique was exploited to characterize the HuPrP interaction (in the native and in a pathological mutated form) with a copper ion, overcoming the several hindrances for the obtaining of a NMR or X-ray structure. MD simulations of the models obtained, validated as for HdSOD systems, revealed features suggesting a role for the ion in facilitating misfolding process, according to what previously hypothesized in literature. Our results, together with several others available today, make it clear that the applications of MD will play an even more important role for the understanding of biology in the future.