Solution-based approach to study binding to the eIF4E cap-binding site using CD spectroscopy
Colin W. Garvie
The eukaryotic initiation factor 4E (eIF4E) is the key component of the translational initiation complex that recruits mRNA by binding to a unique “cap” structure located at the 5′ end of the mRNA. Overexpression of eIF4E has been implicated in the development of cancer, potentially as a result of increasing the cellular levels of proteins involved in processes that include proliferation and regulation of apoptosis. As a result, the cap-binding site of eIF4E has become a target for the development of anti-cancer therapeutics. The structure of eIF4E bound to the cap mimic 7-methyl-GDP revealed that two tryptophans from different loops in eIF4E sandwiched the 7-methylguanine group between them. This interaction gives rise to a strong exciton coupling signal between the two tryptophans that can be visualized by CD spectroscopy. eIF4E is a challenging protein to work with because of a propensity to aggregate under conditions used in biophysical techniques. CD spectroscopy provides a gentle, solution-based approach to study binding to the cap-binding site of eIF4E. Evidence is provided that the exciton coupling signal can be used to both qualitatively and quantitatively analyze the binding of cap analogs to eIF4E.
Circular dichroism, Secondary structure, Protein denaturation, Protein folding, Ligand binding, Biochemistry