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

[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.

[学科分类] 生物化学/生物物理

[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