Binding of RPE65 Fragments to Lipid Monolayers and Identification of Its Partners by Glutathione <i>S</i>-Transferase Pull-Down Assays
Bibliographic record
Abstract
RPE65 is the major component of the retinal pigment epithelium (RPE) microsomal membrane, and it plays a critical role in the binding of retinoids involved in the visual cycle. To understand how RPE65 binds to membranes, we have expressed and purified soluble fragments of human RPE65 fused to glutathione S-transferase (GST). The interaction between two fragments of RPE65 (F1 and F2 which include residues 1-125 and 126-250, respectively) and lipid monolayers has been studied by surface pressure, ellipsometry, and surface rheology measurements. Surface pressure and ellipsometry clearly showed a rapid adsorption of F2 to lipid monolayers whereas the kinetics of binding of F1 was much slower. Furthermore, the data suggest that the F2 fragment inserts into the lipid monolayer. Surface rheology showed a clear increase in monolayer rigidity only in the presence of F2, thereby demonstrating high intermolecular interactions of this fragment. This observation is further supported by the GST pull-down assays which demonstrated that F2 cosediments with full-length RPE65, suggesting that RPE65 has the propensity to form clusters or oligomers. The structure homology modeling of RPE65 based on a related family member, apocarotene 15',15'-oxygenase, further suggests that a hydrophobic patch located in the F2 region might be responsible for membrane binding. The present work shows that F2 interacts much stronger with lipid monolayers than does F1, which suggests that the region of RPE65 located between residues 126-250 should be very important for its membrane binding. Moreover, given that these fragments are not acylated, these data also suggest that an effective binding of RPE65 to membranes can be achieved without palmitoylation. Furthermore, GST pull-down assays also indicated that F2 interacts with 11-cis-retinol dehydrogenase, which supports previous data suggesting that it could act as a partner of RPE65.
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How this classification was reachedexpand
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 itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".