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Drug transport mechanism of P-glycoprotein monitored by single molecule fluorescence resonance energy transfer | Stefan Ernst
; Brandy Verhalen
; Nawid Zarrabi
; Stephan Wilkens
; Michael Boersch
; | Date: |
1 Feb 2011 | 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. | Source: | arXiv, 1102.0113 | Services: | Forum | Review | PDF | Favorites |
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