The mRNA cap-binding protein eIF4E is the limiting factor in the eIF4F translation initiation complex, which mediates the binding of the 40S ribosome to the mRNA. 15N relaxation studies have been used to characterize the backbone dynamics of deuterated eIF4E in a CHAPS micelle for the apoprotein, the m7GDP-bound form, and the dinucleotide (m7GpppA)-bound form, as well as for CHAPS-free eIF4E. Large differences in overall correlation time between the CHAPS-free form (11.8 ns) and samples containing different concentrations of CHAPS (15.9-19.4 ns) indicate that eIF4E is embedded in a large micelle in the presence of CHAPS, with a total molecular weight in the range of 40-60 kDa. CHAPS seems to restrict the mobility of the a2-b3 and a4-b5 loops which are thought to be embedded in the micelle. No significant changes in overall mobility were seen between the m7GDP-bound form, the m7GpppA-bound form, and the apoprotein. Amide hydrogen exchange data indicate the presence of slowly exchanging amides in two surface-exposed helices (a2 and a4), as well as the a4-b5 loop, indicating protection by the CHAPS micelle. The micelle covers the convex side of the protein away from the cap-binding site.
Bibliographical noteFunding Information:
We thank Greg Heffron for assistance with NMR spectroscopy, Jeffrey Peng for providing his analysis programs for relaxation, and Nahum Sonenberg and Anne-Claude Gingras for providing the yeast eIF4E construct and the m7GDP affinity resin. This work was supported in part by the National Institute of Health (G.W.), the National Science Foundation (G.W.), and the Harvard Center for Structural Biology and the Giovanni Armenise-Harvard Foundation for Advanced Scientific Research (G.W.). A.M.M. is a Howard Hughes Medical Institute Predoctoral Fellow. H.M. acknowledges partial support from the Toyobo Biotechnology Foundation.
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