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.

journal_name

FEBS Lett

journal_title

FEBS letters

authors

Randak C,Neth P,Auerswald EA,Assfalg-Machleidt I,Roscher AA,Hadorn HB,Machleidt W

doi

10.1016/s0014-5793(96)01217-3

subject

Has Abstract

pub_date

1996-11-25 00:00:00

pages

97-100

issue

1

eissn

0014-5793

issn

1873-3468

pii

S0014-5793(96)01217-3

journal_volume

398

pub_type

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