ESR spectroscopy using spin-labeled ATP was used to study nucleotide binding to and structural transitions within the multidrug resistance P-glycoprotein, Pgp. Spin-labeled ATP, SL-ATP, with the spin label attached to the ribose was observed to be an excellent substrate analog for Pgp. 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 Pgp was observed with a binding affinity of 366 μM in the presence of Mg²⁺. Binding of SL-ATP to wild-type Pgp in the presence of the transport substrate verapamil resulted in reduction of the protein-bound spin label moiety, most likely due to a conformational transition within Pgp 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 Pgp in which all naturally occurring cysteines were substituted for alanines. Equilibrium binding of SL-ATP to this mutant Pgp resulted in maximum binding of two nucleotides; the binding affinity was 223 μM in the absence and 180 μM in the presence of verapamil. 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 Pgp in its transition state. The use of different phosphate analogs to trap SL-ATP in Pgp catalytic site resulted in different conformations that most likely represent different sub-steps of the catalytic cycle. Titration experiments using the MgATP-vanadate trapped wild-type Pgp demonstrated that the transition state is an asymmetric structure with one nucleotide tightly bound at one site while the other site is open and available for nucleotide binding. Binding of verapamil to the drug binding site of Pgp was shown to consistently affects the nucleotide binding domain throughout the catalytic cycle as the phenomenon of chemical reduction of the spin-label in presence of verapamil was also observed in the transition state. Such a reduction was observed only in the presence of higher-temperature induced molecular motion suggesting that the transition-state is a flexible structure. The use of cis(Z)flupentixol and a new tetramethylrosamine derivative JJH162 demonstrated that different Pgp modulators induced different conformational changes of the nucleotide-binding domains.

Human MRP2 was successfully expressed in the methylotrophic yeast Pichia pastoris. It is the first time that MRP2 expression has been reported in non-polarized cells. The protein is strictly expressed in P. pastoris membrane system and appears to be under-glycosylated. Optimization of the 5’UTR region of MRP2 mRNA resulted in enhanced expression of the protein. MRP2 could be partially purified by Ni²⁺-affinity chromatography and anion-exchange chromatography. The purification of large quantities of the MRP2 protein will allow detailed biochemical and biophysical characterization of this important protein.