Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry
Bibliographic record
Abstract
Exposure of the membrane protein bacteriorhodopsin (BR) to SDS induces partial breakdown of the native conformation. The exact structural properties of this SDS state remain a matter of debate, despite its widespread use in BR folding experiments. The current work employs hydroxyl radical (·OH) labeling in conjunction with mass spectrometry (MS)-based peptide mapping for probing the solvent accessibility of individual BR segments in the presence of SDS. Previous work revealed methionine sulfoxide formation to be the dominant oxidative pathway. Those data suggested extensive unfolding of helices A and D in SDS. Unfortunately, the lack of Met residues in helices C and F implies that no direct information on the behavior of the latter two elements could be obtained. Here, we address this problem by employing two variants with additional Met residues, L93M (helix C) and V179M (helix F). The oxidation behavior of the resulting 11 methionines can be grouped into three categories: (1) extensively labeled both in native BR and in SDS (loop residues M32, M68, and M163); (2) protected in the native state but not in SDS (M20, M118); (3) always protected (M56, M60, M93, M145, M179, M209). These data show that a solvent-inaccessible core is retained in SDS. This core consists of partially intact helices B, C, E, F, and G. The termini of these helices are highly dynamic and/or unraveled, particularly on the cytoplasmic side. Overall, this work demonstrates how the use of engineered ·OH labeling sites can provide insights into structural properties of membrane proteins.
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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 | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.002 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| 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".