Mechanically Untying a Protein Slipknot: Multiple Pathways Revealed by Force Spectroscopy and Steered Molecular Dynamics Simulations
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Bibliographic record
Abstract
Protein structure is highly diverse when considering a wide range of protein types, helping to give rise to the multitude of functions that proteins perform. In particular, certain proteins are known to adopt a knotted or slipknotted fold. How such proteins undergo mechanical unfolding was investigated utilizing a combination of single molecule atomic force microscopy (AFM), protein engineering, and steered molecular dynamics (SMD) simulations to show the mechanical unfolding mechanism of the slipknotted protein AFV3-109. Our results reveal that the mechanical unfolding of AFV3-109 can proceed via multiple parallel unfolding pathways that all cause the protein slipknot to untie and the polypeptide chain to completely extend. These distinct unfolding pathways proceed via either a two- or three-state unfolding process involving the formation of a well-defined, stable intermediate state. SMD simulations predict the same contour length increments for different unfolding pathways as single molecule AFM results, thus providing a plausible molecular mechanism for the mechanical unfolding of AFV3-109. These SMD simulations also reveal that two-state unfolding is initiated from both the N- and C-termini, while three-state unfolding is initiated only from the C-terminus. In both pathways, the protein slipknot was untied during unfolding, and no tightened slipknot conformation was observed. Detailed analysis revealed that interactions between key structural elements lock the knotting loop in place, preventing it from shrinking and the formation of a tightened slipknot conformation. Our results demonstrate the bifurcation of the mechanical unfolding pathway of AFV3-109 and point to the generality of a kinetic partitioning mechanism for protein folding/unfolding.
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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 | 0.000 | 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 it