The eukaryotic initiation factor 3 (eIF3) plays an important role in translation initiation, acting as a docking site for several eIFs that assemble on the 40S ribosomal subunit. Here, we use mass spectrometry to probe the subunit interactions within the human eIF3 complex. Our results show that the 13-subunit complex can be maintained intact in the gas phase, enabling us to establish unambiguously its stoichiometry and its overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Dissociation takes place as a function of ionic strength to form three stable modules eIF3(c:d:e:l:k), eIF3(f:h:m), and eIF3(a:b:i:g). These modules are linked by interactions between subunits eIF3b:c and eIF3c:h. We confirmed our interaction map with the homologous yeast eIF3 complex that contains the five core subunits found in the human eIF3 and supplemented our data with results from immunoprecipitation. These results, together with the 27 subcomplexes identified with increasing ionic strength, enable us to define a comprehensive interaction map for this 800-kDa species. Our interaction map allows comparison of free eIF3 with that bound to the hepatitis C virus internal ribosome entry site (HCV-IRES) RNA. We also compare our eIF3 interaction map with related complexes, containing evolutionarily conserved protein domains, and reveal the location of subunits containing RNA recognition motifs proximal to the decoding center of the 40S subunit of the ribosome.hepatitis C virus internal ribosome entry site ͉ subunit organization model3 ͉ top-down analysis of macromolecular complexes ͉ translation regulation ͉ in-solution disruption S ince its identification in the 1970s (1-3), the translation initiation factor eIF3 has been subjected to intense scrutiny. Despite considerable interest, knowledge of many aspects of its structure and function remain elusive because of its overall structural complexity and the lack of facile genetic approaches. It is established, however, that eIF3 is involved in both ribosome biogenesis and protein synthesis in eukaryotes (4). Concerted binding of initiation factors is required to initiate protein synthesis and recruit transfer and messenger RNAs to the 40S subunit before assembly of active ribosomes (5). eIF3 binding may take place initially during this process, together with eIF1 and eIF1A to the 40S, followed by binding of the Met-tRNA i -eIF2-GTP complex. Then mRNA binding, scanning, and AUG recognition occur, enabling the 60S subunit to join to form elongation-competent 80S ribosomes (6). An alternative pathway of initiating protein synthesis, often used by viruses, involves a structured sequence in the 5Ј untranslated region of mRNA known as the internal ribosome entry site (IRES). These sequences promote translation initiation without requiring the full complement of eukaryotic initiation factors (5-7). The hepatitis C virus (HCV) IRES is recognized specifically by the small ribosomal subunit and eIF3 before viral translation initiation, forming stable complexes ...
The methodology developed in the research presented herein makes use of chaotropic solvents to gently dissociate subunits from an intact macromolecular complex and subsequently allows for the measurement of collision cross section (CCS) for both the recombinant (R-eIF3k) and solvent dissociated form of the subunit (S-eIF3k). In this particular case, the k subunit from the eukaryotic initiation factor 3 (eIF3) was investigated in detail. Experimental and theoretical CCS values show both the recombinant and solvent disrupted forms of the protein to be essentially the same. The ultimate goal of the project is to structurally characterize all the binding partners of eIF3, determine which subunits interact directly, and investigate how subunits may change conformation when they form complexes with other proteins.
Background: Thioethers have been observed in therapeutic antibodies, with increasing levels upon storage. Results: IgG1 thioether bond formation is naturally occurring, but the formation rate depends on light chain type. Conclusion: Slower thioether bond formation on IgG1 is caused by dehydrogenation impairment through its light chain. Significance: Safety concerns associated with thioether control on therapeutic antibodies are diminished by its natural production.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.