A recombinant polypeptide model of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator is a GTP‐binding protein

FEBS Letters

Machleidt, Werner¹  Assfalg-Machleidt, Irmgard¹  Neth, Peter³  Auerswald, Ennes A.²  Hadorn, Hans-Beat³  Randak, Christoph³  Roscher, Adelbert A.³ 

[1] Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Ludwig-Maximilians-Universität, Munich, Germany;Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, Klinikum Innendtadt der Ludwig-Maximilians-Universität, Munich, Germany;Kinderklinik im Dr. von Haunerschen Kinderspital der Ludwig-Maximilians-Universität München, Lindwurmstraβe 4, D-80337 Munich, Germany

关键词: Cystic fibrosis;    Cystic fibrosis transmembrane conductance regulator;    Nucleotide binding;    Guanosine triphosphate;    G-protein;    GTPase activity;    CFTR;    cystic fibrosis transmembrane conductance regulator;    CF;    cystic fibrosis;    ΔF;    fluorescence enhancement;    ΔF m;    maximal fluorescence enhancement (asymptotic maximum of a fitted curve);    GST;    glutathione S-transferase;    GST-NBF-2;    glutathione S-transferase-CFTR-NBF-2 fusion protein;    IPTG;    isopropyl-β-d-thiogalactoside;    NBF;    nucleotide binding fold;    TNP-ATP;    2′;    3′-O-(2;    4;    6-trinitrocyclohexadienylidine) adenosine 5′-triphosphate;    TNP-GTP;    2′;    3′-O-(2;    4;    6-trinitrocyclohexadienylidine) guanosine 5′-triphosphate;   

DOI  :  10.1016/S0014-5793(96)01217-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 K _d value of 3.9 μM. The corrected K _d values for unlabelled guanine nucleotides were determined to be 33 μM for GTP, 92 μM for GDP and 217 μM 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.

【 授权许可】

Unknown