In this PhD thesis computational methods have been employed in order to study different biologically relevant systems. In the first part of the thesis two Ebola virus proteins were studied, namely Viral Protein 24 (VP24) and Viral Protein 35 (VP35), responsible for the inhibition of the immune response . After a brief theoretical introduction to the main computational methods employed in the thesis, a study of VP35 in complex with small organic molecules is presented. These compounds are able to inhibit the interaction between VP35 and viral nucleoprotein. This study confirms the experimental findings highlighting new important key interactions between the protein the inhibitors. Moreover, an Essential Dynamics analysis points out an interesting collective motion of the apo-form that is hindered by the presence of the ligands. Afterwards, the protein-protein interaction VP24-Karyopherin (KPNA) is studied. An atomistic analysis of the interactions at the interface leads to the design of a nonapeptide with VP24 binding capability. The peptide is derived from a KPNA subsequence and could potentially inhibit the VP24-KPNA interaction. Subsequently an analysis on the pockets present on VP24 surface in different solvents is performed. Once the most promising pocket has been located, a virtual screening on a subset of ZINC database is carried out, leading to the identification of few classes of molecules potentially able to bind VP24. Finally the effect of the osmolytes on VP24 protein structure is studied, pointing out how osmoprotectants and urea have opposite effects on the protein, the former stabilizing the folded state and the latter shifting the equilibrium to the denatured state. In the second part of the manuscript the study of the interaction of an antimicrobial peptide with a lipid membrane is presented. This work was carried out in the University of Groningen under the supervision of Prof. Siewert Jan Marrink in order to deepen the Coarse Grain method. Advisors/Committee Members: tutor: M. Sironi, co-tutor: S. Pieraccini, coordinatore: E. Licandro, LICANDRO, EMANUELA.

In protein synthesis, a key component of the cellular machinery is transfer RNA (tRNA). This small nucleic acid is crucial to the maintenance of the genetic code because it discriminately binds the messenger RNA codon at the ribosome and adds the cognate amino acid to the growing polypeptide chain. The role of tRNA as an adaptor molecule has been understood for decades, but details about the charging of tRNA with cognate amino acids prior to entering the ribosome are still emerging. Aminoacyl-tRNA synthetases (aaRSs) are enzymes that recognize specific tRNAs and amino acids from the cellular pool and facilitate the charging of the correct amino acids on tRNAs. Following aminoacylation, tRNAs dissociate from the aaRSs and bind the elongation factor Tu (EF-Tu) for delivery to the ribosome. The recognition of specific tRNA species by the aaRSs, EF-Tu, and other enzymes along the translation pathway is based on sets of highly conserved nucleotides within different groups of tRNA species. Previous work to identify these recognition elements has focused on experimental studies of single organisms. Here, bioinformatic analyses are used to predict recognition elements for groups of tRNA organized by domain of life and specificity. Shannon entropy differences between evolutionary profiles of tRNA domain/specificity groups and the representatives of all tRNA species reveal the uniquely conserved nucleotides within each tRNA domain/specificity, consistent with experiment. Comparative analysis of consensus sequences for these evolutionary profiles is used to locate tuning elements, also consistent with experiment. The discriminator base and the G53:C63 base pair are identified as conserved in several tRNA domain/specificities, particularly among Archaea. Both sets of predictions expand on the current knowledge of recognition elements, providing suggestions for new mutation studies. AaRS:tRNA complex formation and the aminoacylation reaction have been well-characterized through many high resolution crystal structures and biochemical assays, but dissociation of the charged tRNA with subsequent binding to EF-Tu is not well understood. Using molecular modeling and molecular dynamics simulations, the effects of protonation states and the presence/absence of substrates and EF-Tu on tRNA release are explored. Using multiple dynamics and energetics analyses, the migration of protons from the 3' end of the tRNA and the alpha-ammonium group on the charging amino acid is shown to accelerate tRNA dissociation. The presence of AMP has only a minimal effect. Further, pKa calculations predict that Glu41, a conserved residue binding the alpha-ammonium group of the charging amino acid, is part of a proton relay system for releasing the charging amino acid upon transfer. This system is conserved both in structure and sequences across homologous aaRSs and may represent a universal handle for binding and releasing the charging amino acid. Addition of EF-Tu to the aaRS:tRNA complex stimulates tRNA dissociation. Knowledge of the… Advisors/Committee Members: Luthey-Schulten, Zaida A. (advisor), Luthey-Schulten, Zaida A. (Committee Chair), van der Donk, Wilfred A. (committee member), Mitchell, Douglas A. (committee member), Tajkhorshid, Emad (committee member).