Calcium-Induced Structural Transitions of the Calmodulin−Melittin System Studied by Electrospray Mass Spectrometry: Conformational Subpopulations and Metal-Unsaturated Intermediates
Bibliographic record
Abstract
Calmodulin (CaM) is a calcium-sensing protein that can bind to and activate various target enzymes. Here, electrospray ionization mass spectrometry (ESI-MS) was used to investigate calcium-induced structural changes of CaM, as well as binding to the model target melittin (Mel). Nonspecific metalation artifacts were eliminated by conducting the experiments in negative ion mode and with calcium tartrate as metal source [Pan et al. (2009) Anal. Chem. 81, 5008]. Two coexisting CaM subpopulations can be distinguished on the basis of their ESI charge state distributions, namely, relatively disordered conformers (CaM(D), high charge states) and more tightly folded proteins (CaM(F), low charge states). Calcium titration experiments on isolated CaM reveal that the transition from apo-CaM(D) to Ca(4).CaM(F) proceeds with apparent K(d) values of 10, 14, 30, and 12 microM. In the presence of Mel, a gradual [Ca(2+)] increase results in an overall population shift from apo-CaM(D) to Ca(4).CaM(F).Mel. This transition involves various intermediates, Ca(n).CaM(F).Mel with n = 0, ..., 3, as well as apo-CaM(D).Mel. Thus, neither the binding of four Ca(2+) nor the existence of a tightly folded CaM conformation is a prerequisite for target binding. Millisecond time-resolved ESI-MS experiments were conducted to monitor the response of a premixed CaM-Mel solution to a calcium concentration jump, thereby mimicking the conditions encountered in a cellular signaling context. The resulting data suggest that the formation of Ca(4).CaM(F).Mel proceeds along three parallel kinetic pathways: (1) metal binding to CaM(D) followed by formation of a compact protein-target complex, (2) folding of the apoprotein, then target binding, followed by metal complexation, (3) target binding to apo-CaM(D) followed by sequential metal binding. The exact structural properties of the various metal-unsaturated CaM species, as well as their physiological roles, remain to be elucidated.
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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 | 0.000 | 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 | 0.001 |
| 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 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".