Drug transport mechanism of P-glycoprotein monitored by single molecule fluorescence resonance energy transfer

TL;DR

This study uses single-molecule FRET to investigate the catalytic mechanism of P-glycoprotein, revealing its dynamic conformational changes during ATP hydrolysis and drug transport, which enhances understanding of its function in drug resistance.

Contribution

It introduces a novel application of single-molecule FRET with site-specific labeling to monitor Pgp's conformational dynamics during catalysis.

Findings

Pgp fluctuates between at least two major conformations during activity

Significant movements of nucleotide binding domains occur during ATP hydrolysis

FRET efficiency changes correlate with catalytic states

Abstract

In this work we monitor the catalytic mechanism of P-glycoprotein (Pgp) using single-molecule fluorescence resonance energy transfer (FRET). Pgp, a member of the ATP binding cassette family of transport proteins, is found in the plasma membrane of animal cells where it is involved in the ATP hydrolysis driven export of hydrophobic molecules. When expressed in the plasma membrane of cancer cells, the transport activity of Pgp can lead to the failure of chemotherapy by excluding the mostly hydrophobic drugs from the interior of the cell. Despite ongoing effort, the catalytic mechanism by which Pgp couples MgATP binding and hydrolysis to translocation of drug molecules across the lipid bilayer is poorly understood. Using site directed mutagenesis, we have introduced cysteine residues for fluorescence labeling into different regions of the nucleotide binding domains (NBDs) of Pgp. Double-labeled single Pgp molecules showed fluctuating FRET efficiencies during drug stimulated ATP hydrolysis suggesting that the NBDs undergo significant movements during catalysis. Duty cycle-optimized alternating laser excitation (DCO-ALEX) is applied to minimize FRET artifacts and to select the appropriate molecules. The data show that Pgp is a highly dynamic enzyme that appears to fluctuate between at least two major conformations during steady state turnover.