Biophysical approach to studies of cap-eIF4E interaction by synthetic cap analogs.

Specific recognition of mRNA 5' cap by eukaryotic initiation factor eIF4E is a rate-limiting step in the translation initiation. Structural determination of the eIF4E-cap complexes, as well as complexes of eIF4E with other proteins regulating its activity, requires complementary experiments that allow for energetic and dynamic aspects of formation and stability of the complexes. Such a combined approach provides information on the binding mechanisms and, hence, may lead to mechanistic models of eIF4E functioning and regulation on the molecular level. This chapter summarizes in detail the method of experiments used to probe the cap-binding center of eIF4E, steady state and stopped-flow fluorescence, and microcalorimetry. The studies were performed with a wide class of synthetic, structurally modified cap analogs that resembles in some respect an application of site directed mutagenesis of the protein. The chapter presents a general recipe as to how to investigate protein-ligand interactions if the protein has no enzymatic activity and both the protein and the ligand absorb and emit UV/VIS radiation in the same spectral ranges.