Characterization of Transient Protein Folding Intermediates during Myoglobin Reconstitution by Time-Resolved Electrospray Mass Spectrometry with On-Line Isotopic Pulse Labeling
Bibliographic record
Abstract
A novel technique for studying protein folding kinetics is presented. It is based on a continuous-flow setup that is coupled to an electrospray (ESI) mass spectrometer and allows initiation of a folding reaction, followed by isotopic pulse labeling. The protein is electrosprayed "quasi-instantaneously" after exposure to the deuterated solvent. This approach yields structural information from the ESI charge state distribution and from the H/D exchange levels of individual protein states, while at the same time noncovalent interactions can be monitored. This technique is used to study the reconstitution of holomyoglobin (hMb) from unfolded apomyoglobin (aMb) and free heme. MS/MS is used to establish that a short-lived folding intermediate with two heme groups attached represents a protein-bound heme dimer. This state appears to have a compactness close to that of native hMb; however, isotopic labeling indicates a significantly perturbed structure. Another intermediate is bound to a single heme group and shows a charge state distribution similar to that of unfolded aMb. Exchange levels exhibited by this state are lower than for unfolded aMb, indicating that fewer hydrogens are exposed to the solvent and/or that more of them are involved in hydrogen bonding. Native hMb leads to the formation of low charge state ions (hMb(9+), hMb(8+)) and shows low exchange levels. However, early during reconstitution, a slightly unfolded form of the heme-protein complex contributes to the observed hMb(9+) ions. A peak width analysis reveals that the structural heterogeneity of some of the observed protein species decreases as reconstitution proceeds.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| 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 source (direct Gemma or distilled Codex), 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".