Studying tertiary structural transitions of tRNALys3 and Arf1 protein with pressure

Abstract

RNA is believed to be one of the original molecular components present in the beginning of life about 3.5 billion years ago. RNA structure comprises both double helical secondary structures involving canonical nucleic acid base pairing and stacking, as well as tertiary structures in which more sequence distant interactions fold the molecules into a more compact structure. The molecular determinants of folded RNA structures and the pathways of their folding remain poorly understood. First half of this dissertation focuses on the effects of pressure perturbation on structural transitions of RNA molecules. According to Le Chatelier’s principle, pressure shifts (bio)chemical equilibria towards states of lower molar volume. The differences in volume between biomolecular conformations can arise from differences in packing and cavities, differences in interaction with solvent molecules, including electrostriction for charged molecules such as RNA. The effect of pressure on the structure of a folded RNA molecule, tRNALys3, was investigated using a combination of Nuclear Magnetic Resonance (NMR) and Small Angle X-ray Scattering (SAXS). Pressure leads to significant perturbation of the local interactions that define the tRNA tertiary structure, with little effect on secondary structure. Moreover, the global shape of the molecule was not strongly affected by pressure. The effects of divalent cation and water activity on these transitions were explored as well. In the second half of this dissertation, study model has changed from RNA to protein that we studied on one of the small GTPases - ADP ribosylation factors (Arfs). Arfs function in regulation of vesicular transport with lipids and protein trafficking in eukaryotic cells. GTPase activity of Arfs is activated by Guanine nucleotide Exchange Factors (GEFs); then inactivated upon binding GTPase Activating Proteins (GAPs). As a GDP/GTP switch, massive conformational differences between the GDP- and GTP-bound forms are observed in the N-terminal and switch regions of Arfs. In addition, previous studies suggest the N-terminal helix controls the conformational transitions of GDP/GTP switch that involves local unfolding. Using pressure perturbation coupled with NMR and SAXS, we have characterized the excited conformational states likely populated during the switch transitions. In addition, we have investigated the effects of GDP and Mg2+ on the Arf1 conformational landscape. These results helped to map a plausible GDP/GTP switch pathway and better understand the allosteric mechanism of Arfs family of small GTPases.