Recombinant synthesis, purification, and nucleotide binding characteristics of the first nucleotide binding domain of the cystic fibrosis gene product.
The majority of mutations which lead to clinical cystic fibrosis are located within the two predicted nucleotide binding domains of the cystic fibrosis gene product. We have used a prokaryotic expression system to synthesize and purify the first nucleotide binding domain (NBD-1, amino acids 426-588) with and without the most common mutation associated with the disease (the deletion of phenylalanine at position 508, delta F508). Both wild type and delta F508 NBD-1 bind ATP-agarose in a quantitatively comparable manner; this binding was inhibited by excess Na2ATP, trinitrophenol-ATP, or 8-azido-ATP. Irreversible NBD-1 labeling by an ATP analog was demonstrated using [32P]8-azido-ATP. This covalent labeling was inhibited by preincubation with Na2ATP, with half-maximal inhibition for Na2ATP occurring at approximately 5 mM for both the wild type and delta F508 nucleotide binding domain. These experiments are among the first to confirm the expectation that the cystic fibrosis transmembrane conductance regulator NBD-1 binds nucleotide. Since, under the conditions used in our study, NBD-1 without phenylalanine 508 displays very similar nucleotide binding characteristics to the wild type protein, our results support previous structural models which predict that the delta F508 mutation should not cause an alteration in ATP binding.
Gregory Fleming James Cystic Fibrosis Research Center, Department of Medicine, School of Medicine, University of Alabama, Birmingham 35294.
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000345:Affinity Labels; D001386:Azides / Q000737:chemistry; D001483:Base Sequence; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics
D000345:Affinity Labels; D001386:Azides; C505032:CFTR protein, human; D008565:Membrane Proteins; D009711:Nucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D010649:Phenylalanine; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D004247:DNA