Kinetic Unfolding Mechanism of the Inducible Nitric Oxide Synthase Oxygenase Domain Determined by Time-Resolved Electrospray Mass Spectrometry
Bibliographic record
Abstract
The inducible nitric oxide synthase core oxygen domain (iNOS(COD)) is a homodimeric protein complex of ca. 100 kDa. In this work, the subunit disassembly and unfolding of the protein following a pH jump from 7.5 to 2.8 were monitored by on-line rapid mixing in conjunction with electrospray (ESI) time-of-flight mass spectrometry. Various protein species become populated during the denaturation process. These can be distinguished by their ligand binding behavior, and by the different charge states that they produce during ESI. Detailed intensity-time profiles were obtained for all of these species, and the kinetics were subjected to a global analysis which allows a model of the denaturation process to be developed. The data are described well by three relaxation times (tau(1) = 0.36 s, tau(2) = 0.62 s, and tau(3) = 3.3 s), each of which has a characteristic amplitude spectrum. The initial step of the reaction is the disruption of the iNOS(COD) dimer, to generate heme-bound monomeric species in various degrees of unfolding. This first step is accompanied by the loss of two tetrahydrobiopterin cofactors. Subsequent heme loss generates monomeric apoproteins exhibiting various degrees of unfolding. In addition, the formation of proteins that are bound to two heme groups is observed. A subpopulation of holo monomers undergoes substantial unfolding while retaining contact with the heme cofactor. Together with previous studies, the results of this work suggest that the occurrence of complex reaction mechanisms involving several short-lived intermediates is a common feature for the denaturation of large multiprotein complexes.
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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 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.004 | 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".