Intrinsic Fluorescence of the P-glycoprotein Multidrug Transporter: Sensitivity of Tryptophan Residues to Binding of Drugs and Nucleotides
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Bibliographic record
Abstract
P-glycoprotein is a member of the ATP binding cassette family of membrane proteins, and acts as an ATP-driven efflux pump for a diverse group of hydrophobic drugs, natural products, and peptides. The side chains of aromatic amino acids have been proposed to play an important role in recognition and binding of substrates by P-glycoprotein. Steady-state and lifetime fluorescence techniques were used to probe the environment of the 11 tryptophan residues within purified functional P-glycoprotein, and their response to binding of nucleotides and substrates. The emission spectrum of P-glycoprotein indicated that these residues are present in a relatively nonpolar environment, and time-resolved experiments showed the existence of at least two lifetimes. Quenching studies with acrylamide and iodide indicated that those tryptophan residues predominantly contributing to fluorescence emission are buried within the protein structure. Only small differences in Stern-Volmer quenching constants were noted on binding of nucleotides and drugs, arguing against large changes in tryptophan accessibility following substrate binding. P-glycoprotein fluorescence was highly quenched on binding of fluorescent nucleotides, and moderately quenched by ATP, ADP, and AMP-PNP, suggesting that the site for nucleotide binding is located relatively close to tryptophan residues. Drugs, modulators, hydrophobic peptides, and nucleotides quenched the fluorescence of P-glycoprotein in a saturable fashion, allowing estimation of dissociation constants. Many compounds exhibited biphasic quenching, suggesting the existence of multiple drug binding sites. The quenching observed for many substrates was attributable largely to resonance energy transfer, indicating that these compounds may be located close to tryptophan residues within, or adjacent to, the membrane-bound domains. Thus, the regions of P-glycoprotein involved in nucleotide and drug binding appear to be packed together compactly, which would facilitate coupling of ATP hydrolysis to drug transport.
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Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from it