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Kinetics of the association/dissociation cycle of an ATP-binding cassette nucleotide-binding domain.

ABSTRACT: Most ATP binding cassette (ABC) proteins are pumps that transport substrates across biological membranes using the energy of ATP hydrolysis. Functional ABC proteins have two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is unresolved. This is due in part to the limited kinetic information on NBD association and dissociation. Here, we show dimerization of a catalytically active NBD and follow in real time the association and dissociation of NBDs from the changes in fluorescence emission of a tryptophan strategically located at the center of the dimer interface. Spectroscopic and structural studies demonstrated that the tryptophan can be used as dimerization probe, and we showed that under hydrolysis conditions (millimolar MgATP), not only the dimer dissociation rate increases, but also the dimerization rate. Neither dimer formation or dissociation are clearly favored, and the end result is a dynamic equilibrium where the concentrations of monomer and dimer are very similar. We proposed that based on their variable rates of hydrolysis, the rate-limiting step of the hydrolysis cycle may differ among full-length ABC proteins.

SUBMITTER: Zoghbi ME

PROVIDER: S-EPMC3281709 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Kinetics of the association/dissociation cycle of an ATP-binding cassette nucleotide-binding domain.

Zoghbi Maria E ME Fuson Kerry L KL Sutton Roger B RB Altenberg Guillermo A GA

The Journal of biological chemistry 20111209 6

Most ATP binding cassette (ABC) proteins are pumps that transport substrates across biological membranes using the energy of ATP hydrolysis. Functional ABC proteins have two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is unresolved. This is due in part to the limited kinetic information on NBD association and dissociation. Here, we show dimerization of a catalytically active NBD and follow in real time the association and di ...[more]

PMID: 22158619

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Project description:BackgroundIn this study, we evaluated the association between these polymorphisms and gallstone disease using meta-analysis and compared the hepatic ABCG5/G8 mRNA expression and biliary lipids composition in patients with different genotypes of T400K and Y54C.MethodsData were analyzed using the Stata/SE 11.0 software and a random- effects model was applied irrespective of between-study heterogeneity. Hepatic mRNA expression of ABCG5/G8 genes in 182 patients with gallstone disease and 35 gallstone-free patients who underwent cholecystectomy were determined using real-time PCR. Genotypes of Y54C and T400K in the ABCG8 gene were determined by allelic discrimination using either genomic DNA or hepatic cDNA as template by Taqman assays. Biliary compostion in gallbladder bile was assayed in these patients as well.ResultsTen papers including 13 cohorts were included for the final analysis. In the genotype model, the overall association between genotype with gallstone was significant for D19H (OR?=?2.43, 95%CI: 2.23-2.64, P<0.001), and for Y54C (OR?=?1.36, 95%CI: 1.01-1.83, P?=?0.044), or T400K (OR?=?1.17, 95%CI: 0.96-1.43. P?=?0.110). In allele model, minor alleles of D19H polymorphism (allele D: OR?=?2.25, 95%CI: 2.10-2.42, P<0.001) and of T400K polymorphism (allele K: OR?=?1.18, 95%CI: 1.06-1.31, P<0.001) were related with an increased risk of gallstone disease. However, minor allele of Y54C polymorphism (allele Y, OR?=?1.08, 95%CI: 0.96-1.21, P?=?0.146) was not related with gallstone disease. I(2) statistics indicated no significant between-study heterogeneity for all genetic models for any of the three polymorphisms. Funnel plot and Egger's test suggested the absence of publication bias as well. However, no association of T400K and Y54C polymorphism with hepatic ABCG8/G5 mRNA expression or biliary lipids composition was found.ConclusionsOur study showed strong association of D19H polymorphism with gallstone disease. T400K and Y54C polymorphism, though to a less extent, may also relate with gallstone disease.

Dataset Information

Kinetics of the association/dissociation cycle of an ATP-binding cassette nucleotide-binding domain.

Publications

Kinetics of the association/dissociation cycle of an ATP-binding cassette nucleotide-binding domain.

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