Pulsed Hydrogen Exchange and Electrospray Charge-State Distribution as Complementary Probes of Protein Structure in Kinetic Experiments: Implications for Ubiquitin Folding
Bibliographic record
Abstract
The possible involvement of "hidden" kinetic intermediates in the apparent two-state folding of some proteins is currently a matter of debate. This study uses time-resolved electrospray ionization (ESI) mass spectrometry with on-line pulsed hydrogen-deuterium exchange (HDX) for monitoring the refolding of acid/methanol-denatured ubiquitin. It is demonstrated that the ESI charge-state distribution (CSD) and the extent of HDX represent nonredundant probes of the protein structure in solution. When considered in isolation, the data provided by both of these probes are consistent with a two-state behavior, involving only denatured ubiquitin D and refolded protein F. However, a careful comparison of the CSD and HDX kinetics reveals the presence of an additional species, exhibiting a CSD like the folded protein but showing non-native HDX characteristics. This kinetic intermediate, D*, is in rapid equilibrium with D, such that the overall reaction is consistent with the mechanism D <--> D* --> F. The results of this work suggest that the occurrence of transient intermediates may be more widespread than commonly thought, especially in cases where a cursory analysis indicates two-state behavior.
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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) | 0.001 | 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".