Stepwise copper(<scp>i</scp>) binding to metallothionein: a mixed cooperative and non-cooperative mechanism for all 20 copper ions
Why this work is in the frame
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Bibliographic record
Abstract
Copper is a ubiquitous trace metal of vital importance in that it serves as a cofactor in many metalloenzymes. Excess copper becomes harmful if not sequestered appropriately in the cell. As a metal ion chaperone, metallothionein (MT) has been proposed as a key player in zinc and copper homeostasis within the cell. The underlying mechanisms by which MT sequesters and transfers copper ions, and subsequently achieves its proposed biological function remain unknown. Using a combination of electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and emission spectroscopy, we report that the Cu(i) to human apo-MT1a binding mechanism is highly pH-dependent. The 20 relative Kf-values for the binding of 1 to 20 Cu(i) to the 20 cysteines of MT were obtained from computational simulation of the experimental mass spectral results. These data identified the pH-dependent formation of three sequential but completely different Cu–SCYS clusters, as a function of Cu(i) loading. These data provide the first overall sequence for Cu(i) binding in terms of domain specificity and transient binding site structures. Under cooperative binding at pH 7.4, a series of four clusters form: Cu4SCYS-6, followed by Cu6SCYS-9 (β), then a second Cu4SCYS-6 (α), and finally Cu7SCYS-x (α) (x = up to 11). Upon further addition of Cu(i), a mixture of species is formed in a non-cooperative mechanism, saturating the 20 cysteines of MT1a. Using benzoquinone, a cysteine modifier, we were able to confirm that Cu6SCYS-9 formed solely in the N-terminal β-domain, as well as confirming the existence of the presumed Cu4SCYS-6 cluster in the α-domain. Based on the results of ESI-MS and computational simulation we were able to identify Cu:MT speciation that resulted in specific emission and CD spectral properties. Speciation resulting from 20 bimolecular, sequential reactions involving 20 Cu(i) ions binding to metallothionein at different pH-values is reported.
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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 | 0.003 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.002 | 0.000 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 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 it