FRET Analysis Indicates That the Two ATPase Active Sites of the P-Glycoprotein Multidrug Transporter Are Closely Associated
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Bibliographic record
Abstract
Members of the ABC superfamily carry out the transport of various molecules and ions across cellular membranes, powered by ATP hydrolysis. Substantial evidence indicates that the two catalytic sites of the nucleotide binding domains function in a highly cooperative, alternating sites mode, which suggests the possibility that they interact with each other physically. In this study, fluorescence energy transfer experiments were used to estimate the distance between two fluors, each covalently linked to a highly conserved Cys residue (Cys428 and Cys1071) within the Walker A motif of the catalytic site. The vanadate.ADP.Mg(2+) complex was trapped in one catalytic site of membrane-bound or highly purified P-glycoprotein, and the other site was labeled with MIANS [2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid]. Following loss of the trapped vanadate complex, the newly vacant site was then labeled with NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole). The fluorescence properties of the singly labeled P-glycoproteins showed that no energy transfer occurred between MIANS (the donor) and NBD (the acceptor) when they were simply mixed together. On the other hand, the fluorescence emission of the MIANS group in doubly labeled P-glycoprotein was highly quenched as a result of energy transfer to NBD, leading to an estimate of a donor-acceptor separation distance of approximately 16 A for P-glycoprotein labeled in the native plasma membrane and approximately 22 A for P-glycoprotein labeled in detergent solution. The separation of the two fluorophores is compatible with the recently reported crystal structure of the Rad50cd dimer, but not with that of the HisP dimer. These results suggest that the two catalytic sites of the P-glycoprotein nucleotide binding domains are relatively close together, which would facilitate cooperation between them during the catalytic cycle.
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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 | 0.001 |
| Bibliometrics | 0.000 | 0.001 |
| 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