Structural Study of eIF Complexes by H/D Exchange FT-ICR Mass Spectrometry

Abstract : The eukaryotic initiation factor 3 (eIF3) complex plays a core role in the interaction network among several eIFs that assemble on the 40S ribosomes and participate in the different reactions throughout the translation initiation pathway. The Saccharomyces cerevisiaea eIF3 complex comprises five subunits, all of which are the core subunits of the mammalian eIF3 complex consisting of 13 subunits. Attempts to decipher its tridimensionnal structure are under way. A first path to study the structure of this complex is to complete the identification of binding regions, few of which are currently known. Recently, the interaction region between eIF3i and extreme C-terminal domain of eIF3b has been obtained through NMR and crystal structure. On the other hand, the interaction region between 3i and 3g, although located to the N-terminal domain of 3g still remains to be defined. Hydrogen/deuterium exchanges (HDX) have been developed for a long time and are widely used for structural studies of proteins and multiprotein complexes. It is commonly analyzed using mass spectrometry. The most classic standard HDX-MS approach consists in making a mass measurement of deuterium-labelled peptides from an enzymatic digestion of the protein of interest to determine the level and rate of deuterium incorporation. In this study, a high performance 7 T FT-ICR mass spectrometer was used in combination with nanoLC separation to acquire highly accurate HDX-MS data. The precision on the mass measurement of FT-ICR MS is by itself not sufficient to unambiguously identify peptides from a pepsin digest due to the lack of pepsin specificity. We therefore developed a statistical approach for peptide identification, based on a probability of occurrence value of a given peptide within a pepsin digest. In combination with high mass accuracy, this method allows efficient identification of the peptides, without additional need of MS/MS verification. This method has been applied on the study of the binding regions in the complexes of eIF3i:bC3 and eIF3i:gC1ΔC. Peptide reference lists with high sequence coverage and rich sequence superposition ensured structure elucidation with high spatial resolution. For the binding of 3i and 3b, the detailed interaction regions were unveiled for proteins in the solution phase which resembled the physiological condition and were coherent with the reported protein structure, thus provided complimentary information to the crystallographic structure in solid phase. For the binding of 3i and 3g, the interaction regions were studied with the absence of any atomic structural information of 3g. This provides significant insights of the complex formation of 3i and 3g, and for the first time the precise binding regions were successfully revealed.
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Submitted on : Wednesday, December 4, 2013 - 5:25:45 PM
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Jianqing Wu. Structural Study of eIF Complexes by H/D Exchange FT-ICR Mass Spectrometry. Analytical chemistry. Ecole Polytechnique X, 2013. English. ⟨pastel-00914013⟩

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