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Related Concept Videos

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The pharmacogenetics of drug transporters is increasingly recognized as a critical factor influencing interindividual variability in drug absorption, distribution, and elimination. These membrane-bound proteins regulate drugs' movement across cellular barriers by actively pumping them out (efflux) or facilitating their uptake (influx). Among the major transporter families, ATP-binding cassette (ABC) and solute carrier (SLC) transporters play particularly prominent roles. Genetic polymorphisms...
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Related Experiment Video

Updated: May 15, 2026

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase
09:31

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Published on: September 26, 2020

P-glycoprotein is fully active after multiple tryptophan substitutions.

Douglas J Swartz1, Joachim Weber, Ina L Urbatsch

  • 1Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, USA.

Biochimica Et Biophysica Acta
|December 25, 2012
PubMed
Summary

Tryptophan residues in P-glycoprotein (Pgp) are crucial for its function in multidrug resistance. Specific membrane-bound tryptophans maintain drug binding sites, while intracellular tryptophans facilitate communication, enabling functional Pgp mutants with reduced fluorescence.

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Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET
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Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET

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Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence
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Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Published on: April 27, 2018

Related Experiment Videos

Last Updated: May 15, 2026

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase
09:31

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Published on: September 26, 2020

Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET
12:07

Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET

Published on: October 9, 2021

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence
11:51

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Published on: April 27, 2018

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cancer Research

Background:

  • P-glycoprotein (Pgp) is a key efflux pump contributing to multidrug resistance in cancer.
  • Pgp possesses eleven conserved tryptophan (Trp) residues, suggesting their functional importance.
  • Understanding the role of individual Trp residues is critical for developing strategies to overcome Pgp-mediated drug resistance.

Purpose of the Study:

  • To investigate the functional significance of individual tryptophan residues in P-glycoprotein.
  • To determine the specific roles of membrane-bound versus intracellular tryptophans in Pgp activity.
  • To engineer Pgp mutants with altered properties, such as reduced intrinsic fluorescence, while retaining function.

Main Methods:

  • Site-directed mutagenesis was used to replace each of the eleven native tryptophans in Pgp with conservative substitutions.
  • Functional assays were performed in Saccharomyces cerevisiae to assess drug resistance and mating efficiencies of Trp mutants.
  • Purification of active Pgp mutants and characterization of their ATPase activity and drug binding affinities were conducted.
  • Intrinsic tryptophan fluorescence and drug-induced fluorescence quenching were measured to assess conformational changes.

Main Results:

  • Individual Trp mutations at the membrane surface (W44R, W208Y, W132Y, W704Y, W851Y) significantly reduced drug resistance and/or mating efficiency.
  • Mutations in intracellular coupling helices (W158F, W799F) abolished mating but retained most drug resistance.
  • Mutations within the membrane (W228F, W311Y), at the interface (W694L), and in NBD2 (W1104Y) retained high activity.
  • Combined mutants (pair and quadruple) were fully active, purifiable, and exhibited wild-type-like ATPase activity and drug binding.
  • Combined mutations reduced intrinsic fluorescence by 35% without altering drug-induced quenching.

Conclusions:

  • Tryptophan residues at the membrane surface are critical for maintaining the integrity of Pgp's drug-binding sites.
  • Tryptophan residues in the coupling helices are essential for proper interdomain communication in Pgp.
  • Functional Pgp mutants with significantly reduced intrinsic fluorescence can be generated by combining specific Trp mutations, while preserving drug polyspecificity.