Functional nucleotide-binding domain in the F0F1-ATPsynthase alpha subunit from the yeast Schizosaccharomyces pombe.
The segment R165-T330 of the alpha subunit of Schizosaccharomyces pombe F1-ATPase, corresponding to a putative nucleotide-binding domain by comparison with related nucleotide-binding proteins, has been overexpressed in Escherichia coli. Produced as a nonsoluble material, it was purified in a nonnative form, using a rapid procedure that includes one reversed-phase chromatography step. Refolding of the domain, called DN alpha 19, was achieved quantitatively by using a high-dilution step and monitored by circular dichroism and intrinsic fluorescence. Once folded, DN alpha 19 was highly soluble and stable. It bound 1 mol/mol either of adenine or guanine di- or triphosphate nucleotide, with a Kd ranging from 2.3 to 5.4 microM, or of methylanthraniloyl derivatives of the same nucleotides, with a Kd ranging from 0.2 to 0.6 microM. Interesting, DN alpha 19 was able to hydrolyze nucleoside triphosphates at a low but significant rate. The distance between one tryptophan residue located in the nucleotide-binding site and the ribose-linked methylanthraniloyl group of di- or triphosphate nucleotides was estimated by fluorescence resonance energy transfer to be 13 or 11 A, respectively, suggesting that the tryptophan is close to the polyphosphate moiety of the nucleotide. This tryptophan residue was tentatively assigned to W190 by a hydrophobic cluster comparison with the H-ras p21 protein, suggesting that the putative loop of DN alpha 19 containing W190 could play a functional role in nucleotide binding.
Falson
P
P
Centre d'Etudes de SACLAY, Département de Biologie Cellulaire et Moléculaire, Gif sur Yvette, France.
Penin
F
F
Divita
G
G
Lavergne
J P
JP
Di Pietro
A
A
Goody
R S
RS
Gautheron
D C
DC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D017510:Protein Folding; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D009711:Nucleotides; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases