The Folding Mechanism of an Artificial Knotted Protein Characterized by Optical Tweezers

Andrés Bustamante, Maira Rivera, José Molina and Mauricio Baez

Protein folding occurs because establishment of weak interactions observed in the native state (native contacts) predominate over the formation of transient interactions that could promote alternative conformations (non-native contacts). However, knotted proteins defy this view because their polypeptide chains would require to form non-native contacts during its folding. To determine the role of non-native contacts in such topologies, we explored the folding mechanism of an artificial knotted protein (2ouf-knot). In this case, optimization of non-native contacts should play a minor role during its folding. The folding of 2ouf-knot was characterized maintaining the knotted topology in the unfolded state by pulling their C and N extremes by using optical tweezers. The molecular stretching of 2ouf-knot obtained at constant velocity of pulling showed two transitions of unfolding occurring at 9 ± 1 and 14 ± 1 pN, followed by two transitions of refolding at 8 ± 1 and 12 ± 1 pN. This indicates the presence of an intermediate in both directions of the folding reaction of 2ouf-knot. In terms of the molecular extension, the contour length (Lc) associated with the first transition at low force account for the 30% of the expected extension for the fully unfolded polypeptide. Notably, the intermediate of 2ouf-knot was also apparent under equilibrium conditions. At constant force, 2ouf-knot oscillates between the intermediate and the unfolded state extended by 5 and 16 nm with respect to the native state respectively. We observe that there was no full unfolding of the protein without visiting the intermediate state of 5 nm. Therefore, this specie is an obligate step (on-pathway intermediate) for the complete unfolding of the protein. Nevertheless, we also observe that approximately 20% of the times the fully unfolded protein jump until reach a smaller intermediate of 4 nm with very short residence time. This short-live off-pathway intermediates backtrack to the fully unfolded state which refolds to the on-pathway intermediate of 5 nm. These results contrast with the absence of intermediates (on and off-pathway) in the case of a natural knotted protein (MJ0366 of Methanocaldococcus jannaschii) characterized under the same experimental setup. We suggest that the intermediates observed in the case of artificial knots arise by lack of specific non-native contacts that could help to accommodate the knot during unfolding/refolding process. These specific interactions otherwise will help to smooth the energetic landscape of natural knotted proteins.

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