Intrinsic tryptophan fluorescence of rat liver elongation factor eEF-2 to monitor the interaction with guanylic and adenylic nucleotides and related conformational changes.
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.
Sontag
B
B
Institut de Biologie et Chimie des Protéines, CNRS, Lyon, France.
Reboud
A M
AM
Divita
G
G
Di Pietro
A
A
Guillot
D
D
Reboud
J P
JP
D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D000227:Adenine Nucleotides; D006150:Guanine Nucleotides; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D006153:Guanosine Diphosphate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate