Biophysical Studies of Conformational Changes of the Dcp1:Dcp2 mRNA decapping complex

Controlled degradation of messenger RNA (mRNA) is essential for the regulation of gene expression. The removal of the protective 5’ cap structure of eukaryotic mRNA is a central step in this process and it is catalyzed by the Dcp1:Dcp2 decapping complex. During catalysis, Dcp1:Dcp2 undergoes large conformational changes which are regulated by interactions with several activator proteins including Edc1 and Edc3. The exact details of these structural rearrangements and their functional role in the decapping process are not fully understood yet. Nuclear magnetic resonance (NMR) spectroscopy is a well suited method to study dynamic processes and conformational changes of enzymes. In my dissertation I therefore made use of different NMR experiments and complementary techniques to study the mRNA decapping machinery from S. pombe in detail. In Part III, I used 13CH3 methyl group labeling of Dcp2 and conducted methyl Carr–Purcell–Meiboom–Gill (CPMG) NMR relaxation dispersion (RD) experiments on the Dcp1:Dcp2 decapping complex at high hydrostatic pressures. From these experiments I extracted information about the molecular volumes of the different conformations of the complex during the opening-closing pathway and about the effect of ATP on these volumes. This information could not be reliably obtained from known crystal structures, which underlines the potential of pressure-dependent NMR methods to provide insights into structural features of invisible protein conformations, even for high molecular weight complexes. In Part IV, I examined the applicability of 5-fluorotryptophan labeling for studying protein dynamics with 19F NMR experiments in three different biomolecular systems, namely the Dcp1:Dcp2 mRNA decapping complex, the KIX domain of the transcriptional coactivator CREB binding protein and the DcpS scavenger decapping enzyme. I found that the 19F label on the 5-tryptophan has no measureable effect on the backbone dynamics of the examined proteins and slow protein motions could reliably be extracted from 19F NMR experiments that are insensitive to tryptophan side chain motions. In contrast, I found that NMR relaxation experiments on the 19F nucleus in this position do not always allow for the reliable extraction of kinetic and thermodynamic properties, so these data should always be supported by data from complementary techniques. In Part V, I performed single molecule Förster resonance energy transfer (smFRET) experiments on the decapping complex. We first optimized the labeling positions of the fluorophores by testing several different combinations of labeling sites and cross-validated them with NMR experiments of fluorophore labeled Dcp1:Dcp2 complexes. In subsequent smFRET measurements I confirmed previously obtained NMR data and I examined the influence of different cofactors and substrates that are not suited for NMR experiments. Importantly, as the fluorophore labeling can influence the conformational equilibrium of the decapping complex, cross-validation of smFRET data is crucial for a correct interpretation of the FRET efficiencies. In the presence of Edc3, the Dcp1:Dcp2 mRNA decapping complex undergoes liquid-liquid phase separation (LLPS) and maturation into a gel-like state, about which little is still known. To examine changes in conformation and dynamics of Edc3 and Dcp2 in the transition to the mature state, we performed solid state NMR (ssNMR) experiments on mature Edc3 and Dcp1:Dcp2 (Part VI). We construct a model in which, after LLPS, the factors of the mRNA decapping machinery mature into a structurally inhomogeneous, gel-like state in which the molecular motions and intermolecular contacts that are averaged in solution are frozen out and no longer averaged. Our findings demonstrate the great capacity of ssNMR to study large molecular machines in the condensed, phase separated state. Taken together, these results contribute to our knowledge of how conformational changes regulate mRNA decapping and, ultimately, add to a better understanding of how large molecular machines function.