Structural Mass Spectrometry Analysis Of Rna Chaperones And Protein Therapeutics PDF Download

Protein-nucleic acid interactions are paramount for maintaining cellular homeostasis. Characterization of protein-nucleic acid complexes by high-resolution structural biology methods remains a challenge due to intrinsic structural and chemical heterogeneity. Native mass spectrometry (nMS) is a powerful bioanalytical tool for the investigation of proteins and protein complexes; however, it has only sparingly been implemented in the analysis of protein -nucleic acid complexes. This dissertation describes the application of native mass spectrometry to the analysis of RNA and RNA-protein complexes. Chapters 2 and 3 describe the characterization of the stoichiometry of the HIV-1 viral assembly nucleation complex. Using nMS, it was revealed that Gag specifically dimerizes in the presence of RNA containing the HIV-1 packaging signal (Psi), while other RNAs are bound primarily to monomeric Gag. Further investigations focused on the effect of transcription start site heterogeneity on the dimerization of the HIV-1 genomic RNA 5′ untranslated region (5′UTR), and stoichiometry. It was observed that 5′UTRs that begin with a single guanosine preferentially dimerize and are bound by Gag. Chapter 4 focuses on the characterization of a gas-phase separation method (ion mobility) as a structural biology tool for RNA. The effect of magnesium during RNA folding, solution temperature, ionization polarity, and collisional activation on the collision cross section of tRNAPhe were probed. It was observed that magnesium is essential for the folding and stability of tRNAPhe, consistent with previous reports. The collision cross sections (CCS) of tRNAPhe were compared in both positive and negative ionization polarities. The CCS of tRNAPhe refolded in under folding conditions was lower in negative mode relative to positive mode. It was observed that the CCS of WT tRNAPhe was not affected by the solution temperatures tested, however the CCS of a mutant (MT) tRNAPhe, that has a perturbed tertiary interaction network, increased as a function of solution temperature. Furthermore, we also probed the stability of these RNAs using collision-induced unfolding and it was observed that the wild-type RNA underwent collision-induced collapse while the mutant tRNA collapsed to a lesser extent. Lastly, a small dimeric RNA-RNA complex (HJ3) was used to determine whether RNA quaternary structure is preserved upon transfer to the gas phase. The intact dimer was observed in the gas-phase, and surface-induced dissociation was identified as an effective method for probing the stoichiometry and of RNA-RNA complexes. Chapter 5 describes the characterization of Hfq-RNA complex stability and subunit connectivity by surface-induced dissociation and ion mobility. It was observed that the intrinsically disordered C-terminal domains greatly stabilize Hfq. RNA-binding to Hfq destabilizes the wild-type protein yet stabilizes a protein that lacks C-terminal domains. The dissociation products observed for RNA-bound wild-type and the mutant Hfq were remarkably similar, suggesting that the C-terminal domains do not alter the RNA binding interfaces.