Direct Observation of Tug-of-War during the Folding of a Mutually Exclusive Protein
Bibliographic record
Abstract
Although most protein folding studies are carried out on single-domain proteins, over two-thirds of proteins in proteomes are built up from multiple individually folded domains. A significant fraction of these multidomain proteins are domain-insertion proteins, in which one guest domain is inserted into a surface loop of a host protein. Intricate thermodynamic and kinetic coupling between the two domains can have a profound impact on their folding dynamics. Here we use an engineered mutually exclusive protein as a model system to directly illustrate one such complex dynamic process: the "tug-of-war" process during protein folding. By inserting a guest protein I27w34f into a host protein GB1-L5 (GL5), we engineered a novel, mutually exclusive protein, GL5/I27w34f, in which only one domain can remain folded at any given time due to topological constraints imposed by the folded structures. Using stopped-flow techniques, we obtained the first kinetic evidence that the guest and host domains engage in a folding tug-of-war as they attempt to fold, in which the host domain folds rapidly into its three-dimensional structure and is then automatically unfolded, driven by the folding of the guest domain. Our results provided direct evidence that protein folding can generate sufficient mechanical strain to unravel a host protein. Using single-molecule atomic force microscopy, we provide direct evidence for the existence of a conformational equilibrium between the two mutually exclusive conformations. Our results highlight important roles played by the intricate coupling between folding kinetics, thermodynamic stability, and mechanical strain in the folding of complex multidomain proteins, which cannot be addressed in traditional single-domain protein folding studies.
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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 | 0.000 | 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) | 0.000 | 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".