Intrinsic tryptophan fluorescence of rat liver elongation factor eEF-2 to monitor the interaction with guanylic and adenylic nucleotides and related conformational changes

• Published in: Biochemistry (Easton) 1993-03, Vol.32 (8), p.1976-1980
• Main Authors: Sontag, Bruno, Reboud, Anne Marie, Divita, Gilles, Di Pietro, Attilio, Guillot, Dominique, Reboud, Jean Paul
• Format: Article
• Language: English
• Subjects: Adenine Nucleotides - metabolism; Adenosine Diphosphate - metabolism; Adenosine Triphosphate - metabolism; Animals; Biochemistry, Molecular Biology; Biological and medical sciences; Conformational dynamics in molecular biology; Fundamental and applied biological sciences. Psychology; Guanine Nucleotides - metabolism; Guanosine Diphosphate - metabolism; Guanosine Triphosphate - metabolism; Kinetics; Life Sciences; Mathematics; Molecular biophysics; Peptide Elongation Factor 2; Peptide Elongation Factors - chemistry; Peptide Elongation Factors - metabolism; Protein Conformation; Rats; Spectrometry, Fluorescence; Tryptophan
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00059a014

Elongation factor 2 (eEF-2), which contains seven Trp residues, exhibited a tryptophan-characteristic intrinsic fluorescence with maximum excitation at 280 nm and an emission peak centered at 333 nm that suggested a hydrophobic environment of these tryptophans. Upon denaturation with 6 M guanidine hydrochloride, the maximum emission was shifted to 348 nm. Fluorescence quenching studies using acrylamide and iodide confirmed that the Trp residues were mainly buried in the native molecule and indicated an important heterogeneity, the fractional accessible fluorescence (fa) values being 0.50 and 0.25, respectively. Partial quenching of eEF-2 fluorescence by nucleotides proved the existence of an interaction of the factor in the absence of ribosomes, not only with GDP but also with GTP, nonhydrolyzable analogs, GMP, and adenylic, but not cytidylic, nucleotides. Saturating binding plots showed different maximal changes of fluorescence depending upon the nucleotides, from 6.4% with ADP to 24.5% with GDP, and suggested the existence of more than one binding site for each nucleotide. Among all the nucleotides tested, only GTP at saturating concentration modified the fa value obtained with acrylamide (-36%). The possibility that this modification is related to a conformational change of eEF-2 induced by GTP binding is discussed.