当前位置： SCI文献检索 > BIOCHEMISTRY期刊下所有文献 > Nucleotide binding to the multidrug resistance P-glycoprotein as studied by ESR spectroscopy.

Nucleotide binding to the multidrug resistance P-glycoprotein as studied by ESR spectroscopy.

Abstract：

:Electron spin resonance (ESR) spectroscopy using spin-labeled ATP was used to study nucleotide binding to and structural transitions within the multidrug resistance P-glycoprotein, P-gp. Spin-labeled ATP (SL-ATP) with the spin label attached to the ribose, was observed to be an excellent substrate analogue for P-gp. SL-ATP was hydrolyzed in a drug-stimulated fashion at about 14% of the rate for normal ATP and allowed reversible trapping of the enzyme in transition and ground states. Equilibrium binding of a total of two nucleotides per P-gp was observed with a binding affinity of 366 microM in the presence of Mg2+ but in the absence of transport substrates such as verapamil. Binding of SL-ATP to wild-type P-gp in the presence of verapamil resulted in reduction of the protein-bound spin-label moiety, most likely due to a conformational transition within P-gp that positioned cysteines in close proximity to the spin label to allow chemical reduction of the radical. We circumvented this problem by using a mutant of P-gp in which all naturally occurring cysteines were substituted for alanines. Equilibrium binding of SL-ATP to this mutant P-gp resulted in maximum binding of two nucleotides; the binding affinity was 223 microM in the absence and 180 microM in the presence of verapamil. The corresponding ESR spectra of wild-type and Cys-less P-gp in the presence of SL-ATP indicate that a cysteine side chain of P-gp is located close to the ribose of the bound nucleotide. Trapping SL-ATP as an AlF(x)-adduct resulted in ESR spectra that showed strong immobilization of the radical, supporting the formation of a closed conformation of P-gp in its transition state. This study is the first to employ ESR spectroscopy with the use of spin-labeled nucleotide analogues to study P-glycoprotein. The study shows that SL-ATP is an excellent substrate analogue that will allow further exploration of structure and dynamics within the nucleotide binding domains of this important enzyme.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Delannoy S,Urbatsch IL,Tombline G,Senior AE,Vogel PD

doi

10.1021/bi0512445

subject

Has Abstract

pub_date

2005-10-25 00:00:00

pages

14010-9

issue

eissn

0006-2960

issn

1520-4995

journal_volume

pub_type

杂志文章

在线工具

Nucleotide binding to the multidrug resistance P-glycoprotein as studied by ESR spectroscopy.