Investigating the stability of nitric oxide synthase quaternary structure to denaturant and temperature
Bibliographic record
Abstract
A limitation to investigations of homodimeric protein dissociation is that the signals produced from methods such as fluorescence and circular dichroism represent both dissociation and protein unfolding that may be occurring simultaneously within a sample.Although size exclusion chromatography examines the state of a protein's quaternary structure, complicated overlapping peaks representative of oligomer and monomer can result.To address these limitations the mixed dimer system has been adopted to investigate the dissociation of a homodimeric protein.A mixed dimer is a species in which each subunit of a homodimeric protein is associated with a different affinity tag.The two tags used are the His 6 -tag and the Glu 7 -tag.Such a mixed dimer will bind to a metal chelating column such as Ni-NTA so long as the dimer is intact.Denaturant-or temperature induced dimer dissociation can be detected by the amount of Glu 7 -tagged subunit present in the unbound fraction after the protein is passed over an Ni-NTA resin.SDS PAGE and densitometry assess the amount of Glu 7 -tagged subunit present in those unbound fractions.The experimental conditions necessary to implement this method were developed, and then applied to mammalian inducible nitric oxide synthase (iNOS) and Staphylococcus aureus NOS (SaNOS).With respect to both urea and temperature, the stability of SaNOS is higher than that of iNOS in spite of the bacterial enzyme having a much smaller dimer contact surface.We have also used the mixed dimer method to estimate an equilibrium dissociation constant (K D ) for iNOS dissociation of no greater than 2.3M.This value is compared to the results obtained for iNOS by analytical ultracentrifugation, which can characterize protein complexes and their stoichiometry.
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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) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 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".