title: A recombinant polypeptide model of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator is a GTP-binding protein.
abstract: Association reactions of a recombinant CFTR-NBF-2 polypeptide fused to glutathione S-transferase with guanine nucleotides were monitored quantitatively by recording the fluorescence enhancement of excited trinitrophenol (TNP)-labelled GTP after binding to NBF-2. Binding of TNP-GTP to the recombinant NBF-2 polypeptide was characterized by a Kd value of 3.9 microM. The corrected Kd values for unlabelled guanine nucleotides were determined to be 33 microM for GTP, 92 microM for GDP and 217 microM for GMP. TNP-ATP bound to NBF-2 was competitively displaced by GTP indicating a common binding site for both nucleotides. The recombinant NBF-2 did not show an intrinsic GTPase activity above a detection limit of 0.007 min(-1). Our findings provide the first experimental evidence that NBF-2 can act as a GTP-binding subunit that would favor the release of GDP after GTP hydrolysis.
authors.affiliation: Kinderklinik im Dr. von Haunerschen Kinderspital der Ludwig-Maximilians-Universität München, Munich, Germany. randak@clinbio.med.uni-muenchen.de
authors.forename: C
authors.initials: C
authors.lastname: Randak
authors.forename: P
authors.initials: P
authors.lastname: Neth
authors.forename: E A
authors.initials: EA
authors.lastname: Auerswald
authors.forename: I
authors.initials: I
authors.lastname: Assfalg-Machleidt
authors.forename: A A
authors.initials: AA
authors.lastname: Roscher
authors.forename: H B
authors.initials: HB
authors.lastname: Hadorn
authors.forename: W
authors.initials: W
authors.lastname: Machleidt
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D005453:Fluorescence; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006147:Guanine / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006153:Guanosine Diphosphate; D006147:Guanine; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D020558:GTP Phosphohydrolases