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Circular dichroism and fluorescence studies on five mutant forms of protein synthesis initiation factor eIF‐4E, from the yeast Saccharomyces cerevisiae
Author(s) -
McCubbin William D.,
Edery Isaac,
Altmann Michael,
Sonenberg Nahum,
Kay Cyril M.
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)80233-9
Subject(s) - mutant , circular dichroism , tryptophan , quenching (fluorescence) , protein secondary structure , helix (gastropod) , chemistry , fluorescence , nucleotide , saccharomyces cerevisiae , biochemistry , alpha helix , biophysics , crystallography , yeast , biology , amino acid , gene , ecology , physics , quantum mechanics , snail
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 m 7 GpppG 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 m 7 GpppG‐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 m 7 GpppG. 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.