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Internal and overall motions of the translation factor eIF4E: cap binding and insertion in a CHAPS detergent micelle
A M McGuire1, H Matsuo, G Wagner
1Graduate Program in Biophysics, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
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 m7 GDP-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.
Insights
The mRNA cap-binding protein eIF4E
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- The eukaryotic translation initiation factor 4E (eIF4E) is crucial for mRNA binding to ribosomes.
- eIF4E is the rate-limiting component of the eIF4F translation initiation complex.
- Understanding eIF4E's dynamics and interactions is key to regulating protein synthesis.
Purpose of the Study:
- To investigate the backbone dynamics of eIF4E in solution and in the presence of CHAPS micelles.
- To determine how CHAPS micelles affect eIF4E's structure and mobility.
- To assess the impact of ligand binding (m7GDP and m7GpppA) on eIF4E dynamics within micelles.
Main Methods:
- Utilized 15N relaxation studies on isotopically labeled eIF4E.
- Performed experiments on both CHAPS-free and CHAPS-bound eIF4E, including ligand-bound states.
- Employed amide hydrogen exchange measurements to identify protected regions.
Main Results:
- CHAPS micelles significantly increase the overall molecular weight and restrict eIF4E's mobility, indicating micelle embedding.
- Specific loops (a2-b3, a4-b5) and surface helices (a2, a4) are protected by the micelle.
- Ligand binding (m7GDP, m7GpppA) did not alter the overall mobility of eIF4E within the micelle.
Conclusions:
- CHAPS micelles interact with eIF4E, primarily on its convex surface away from the cap-binding site.
- The micelle binding restricts protein dynamics and protects specific structural elements.
- eIF4E's cap-binding function appears independent of micelle-induced mobility changes.