Circular dichroism and fluorescence studies on five mutant forms of protein synthesis initiation factor eIF‐4E, from the yeast Saccharomyces cerevisiae

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Abstract
CD studies have shown that five tryptophan to phenylalanine (W→F) mutants of eukaryotic initiation factor-4E (eIF-4E) contain low amounts of α-helix, the main elements of secondary structure being β-sheets/turns and aperiodic regions. Interactions with the cap analog m7GpppG are accompanied by changes in overall secondary structure which include reductions, and in one case an increase in α-helix content, as well as increases in total β-structure (3 mutant forms) and decreases in total β-structure (2 mutant forms). These changes may also involve more significant perturbations of localized regions containing phenylalanine residues either involved in nucleotide binding, or close to the nucleotide-binding site. Measurements of intrinsic Trp fluorescence have shown different quantum yields and reduced m7GpppG-induced quenching (with one exception). Acrylamide quenching studies yielded similar parameters for 4 of the mutants but 1 form displayed significantly reduced values. Melting experiments showed that the Trp fluorescence of 4 of the mutants decreased as the temperature was increased, this effect being reduced in 3 cases in the presence of m7GpppG. W 58 F showed an increase in fluorescence as the temperature was raised and this effect was accentuated in the presence of nucleotide. A preliminary attempt has been made to correlate the spectroscopic data with the known biological importance of the individual Trp residues.