Structural Characterization of an Integral Membrane Protein in Its Natural Lipid Environment by Oxidative Methionine Labeling and Mass Spectrometry
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Bibliographic record
Abstract
Membrane proteins represent formidable challenges for many analytical techniques. Studies on these systems are often carried out after surfactant solubilization. Unfortunately, such a non-natural protein environment can affect conformation and stability, and it offers only partial protection against aggregation. This work employs bacteriorhodopsin (BR) as a model system for in situ structural studies on a membrane protein in its natural lipid bilayer. BR-containing purple membrane suspensions were exposed to hydroxyl radicals, generated by nanosecond laser photolysis of dilute aqueous H(2)O(2). The experiments rely on the premise that oxidative labeling occurs mainly at solvent-exposed side chains, whereas sites that are sterically protected will react to a much lesser extent. Following .OH exposure, the protein was analyzed by tryptic peptide mapping and electrospray tandem mass spectrometry. Oxidative labeling of BR was found to occur only at its nine Met residues. This is in contrast to the behavior of previously studied water-soluble proteins, which generally undergo modifications at many different types of residues. In those earlier experiments the high reactivity of Met has hampered its use as a structural probe. In contrast, the Met oxidation pattern observed here is in excellent agreement with the native BR structure. Extensive labeling is seen for Met32, 68, and 163, all of which are located in solvent-exposed loops. The remaining six Met residues are deeply buried and show severalfold less oxidation. Our results demonstrate the usefulness of Met oxidative labeling for structural studies on membrane proteins, especially when considering that many of these species are methionine-rich. The introduction of additional Met residues as conformational probes, as well as in vivo structural investigations, represents exciting future extensions of the methodology described here.
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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