Expression and purification of an isotopically labeled aggregation prone inducible nitric oxide synthase calmodulin‐binding protein for use in nuclear magnetic resonance studies
Bibliographic record
Abstract
Nuclear magnetic resonance (NMR) spectroscopy is an efficient method for studying the dynamics and structures of protein‐protein and protein‐peptide complexes. Calmodulin (CaM), a ubiquitous Ca 2+ ‐sensing protein, is able to bind and regulate various intracellular proteins, including the nitric oxide synthase (NOS) enzymes. The investigation of CaM bound to the CaM‐binding region of the inducible nitric oxide synthase (iNOS) isoform of NOS proved to be difficult due to the propensity of the iNOS CaM‐binding domain to aggregate when not bound to CaM. In the present study, an isotopically labeled peptide of the CaM‐binding region of iNOS has successfully been expressed and purified. The peptide identity was verified by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectroscopy and further characterized by NMR spectroscopy. These results demonstrate an efficient approach for the expression and purification of individually stable isotope labeled protein complexes for NMR analysis, even if one partner is prone to aggregation or has very low solubility. This technique can also be used to label a specific type of amino acid or radiolabel all of the amino acids in one of the components participating in a protein–protein or protein–peptide complex.
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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 | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 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 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".