Ligand Migration in the Gaseous Insulin-CB7 Complex—A Cautionary Tale About the Use of ECD-MS for Ligand Binding Site Determination
Bibliographic record
Abstract
Knowledge of the structure of protein-ligand complexes can aid in understanding their roles within complex biological processes. Here we use electrospray ionization (ESI) coupled to a Fourier transform ion cyclotron resonance mass spectrometer to investigate the noncovalent binding of the macrocycle cucurbit[7]uril (CB7) to bovine insulin. Recent condensed-phase experiments (Chinai et al., J. Am. Chem. Soc. 133:8810-8813, 2011) indicate that CB7 binds selectively to the N-terminal phenylalanine of the insulin B-chain. Competition experiments employing ESI mass spectrometry to assess complex formation between CB7 and wild type insulin B-chain vs. a mutant B-chain, confirm that the N-terminal phenylalanine plays in important role in solution-phase binding. However, analysis of fragment ions produced by electron capture dissociation (ECD) of CB7 complexed to intact insulin and to the insulin B-chain suggests a different picture. The apparent gas-phase binding site, as identified by the ECD, lies further along the insulin B-chain. Together, these studies thus indicate that the CB7 ligand migrates in the ESI mass spectrometry analysis. Migration is likely aided by the presence of additional interactions between CB7 and the insulin B-chain, which are not observed in the crystal structure. While this conformational difference may result simply from the removal of solvent and addition of excess protons by the ESI, we propose that the migration may be enhanced by charge reduction during the ECD process itself because ion-dipole interactions are key to CB7 binding. The results of this study caution against using ECD-MS as a stand-alone structural probe for the determination of solution-phase binding sites.
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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 | 0.002 | 0.002 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.003 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
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".