Circular dichroism and fluorescence studies on five mutant forms of protein synthesis initiation factor eIF-4E, from the yeast Saccharomyces cerevisiae.
CD studies have shown that five tryptophan to phenylalanine (W----F) mutants of eukaryotic initiation factor-4E (eIF-4E) contain low amounts of alpha-helix, the main elements of secondary structure being beta-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 alpha-helix content, as well as increases in total beta-structure (3 mutant forms) and decreases in total beta-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.
McCubbin
W D
WD
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Edery
I
I
Altmann
M
M
Sonenberg
N
N
Kay
C M
CM
D000178:Acrylamides; D002942:Circular Dichroism; D039561:Eukaryotic Initiation Factor-4E; D006358:Hot Temperature; D009154:Mutation; D010448:Peptide Initiation Factors / Q000235:genetics; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000032:analysis*; D013050:Spectrometry, Fluorescence
D000178:Acrylamides; D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors