Cystic fibrosis transmembrane conductance regulator: nucleotide binding to a synthetic peptide.
Multiple mutations in the gene responsible for cystic fibrosis are located within a region predicted to encode a nucleotide-binding fold in the amino terminal half of the cystic fibrosis transmembrane conductance regulator protein. A 67-amino acid peptide (P-67) that corresponds to the central region of this putative nucleotide binding site was chemically synthesized and purified. This peptide bound adenine nucleotides. The apparent dissociation constants (Kd's) for the trinitrophenyl (TNP) adenine nucleotides, TNP-adenosine triphosphate, TNP-adenosine diphosphate, and TNP-adenosine monophosphate, were 300 nanomolar, 200 nanomolar, and greater than 1 micromolar, respectively. The Kd for adenosine triphosphate was 300 micromolar. Circular dichroism spectroscopy was used to show that P-67 assumes a predominantly beta sheet structure in solution, a finding that is consistent with secondary structure predictions. On the basis of this information, the phenylalanine at position 508, which is deleted in approximately 70 percent of individuals with cystic fibrosis, was localized to a beta strand within the nucleotide binding peptide. Deletion of this residue is predicted to induce a significant structural change in the beta strand and altered nucleotide binding.
Thomas
P J
PJ
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Shenbagamurthi
P
P
Ysern
X
X
Pedersen
P L
PL
D000227:Adenine Nucleotides / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003550:Cystic Fibrosis / Q000235:genetics* / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data
D000227:Adenine Nucleotides; C505032:CFTR protein, human; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator