Characterization of the conformational changes in recombinant human metallothioneins using ESI-MS and molecular modeling
Bibliographic record
Abstract
Electrospray ionization mass spectrometry (ESI-MS) data and molecular modeling calculations were used to gain mechanistic, conformational, and domain-specific information from the acid-induced demetallation reactions of human metallothionein. The recombinant proteins studied were the single α- and β-rhMT-1a domains and the βα- and αβ-rhMT-1a two-domain species, based on the human metallothionein 1a sequence. Complete molecular models (MM3/MD) for all the fully metallated and demetallated species using a modified force field are reported for the first time. Basic residues that contribute to the ESI-MS charge states are identified from the molecular models. Demetallation took place under equilibrium conditions within a narrow pH range. For the two-domain proteins, these results support a demetallation mechanism involving the initial complete demetallation of one domain followed by the other for both βα-rhMT and αβ-rhMT. Based on the stability of the separate domains, the β domain is predicted to demetallate first in the two-domain rhMTs. Both the α domain and the β domain were observed to bind an excess of one Cd2+ ion. The metallated rhMT structures were shown to have very stable conformations, but only when fully metallated. Two or more conformations were observed at low pH in the ESI-MS data, which are interpreted as arising from the presence of structure, as opposed to a random coil, in the apo-rhMT. This is the first report of the existence of a structure in the two-domain metal-free apo-MT proteins. Only at extremely low pH does the structure open fully to give the highest charge distribution, which is associated with a random coil. Pre-existing structural features in the apo-MT would explain why the metallation reactions occur so rapidly.Key words: recombinant human metallothionein-1 (rhMT1), electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), molecular mechanics/molecular dynamics (MM3/MD).
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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 | 0.000 |
| 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".