Monday, June 12, 2017

Mechanical untying of the smallest knotted protein from different pulling axis using optical tweezers

Maira Rivera, Andrés Bustamante, Yuxin Hao, Rodrigo A Maillard and Mauricio Baez

Knotted polypeptides constitute a group of proteins whose backbone chain entangles in the folded state. The folding mechanism of knotted protein is complex, involving multiple intermediate states. Therefore, it is difficult to determine experimentally the thermodynamic and kinetic barriers associated with threading the polypeptide chain. To address this problem, we used optical tweezers to study the folding mechanism of MJ0366, the smallest protein containing a shallow trefoil knot. Specifically, we mechanically manipulated the knotted protein from different pulling directions to either tighten the knot upon unfolding or to untie the protein from different points of its structure. When the knot was tightened in force-ramp experiments, we observed single unfolding and refolding transitions characterized by unimodal force distributions, and an accompanying change in contour length (Lc = 24 ± 3 nm) consistent with the expected theoretical value. Moreover, in constant-force experiments, we observed that the protein oscillates between two states, whose frequency of interconversion showed a linear dependence with force. These observations suggest a two-state folding mechanism when the trefoil knot remains in the polypeptide chain. However, when the knot is untied by keeping the C-termini free to thread, the protein unfolds in either one or two transitions and at forces higher than those observed when pulling from the N and C termini. Moreover, 50% of the unfolding transitions have a Lc shorter than the expected value, suggesting that the protein can be trapped in a misfolded state during its refolding process. When the knot is untied by keeping both N- and C-termini free to thread, we observed a single unfolding force distribution with the expected Lc for this construct. However, the refolding process could not be characterized because it occurs at forces below the resolution limit (<1 pN). Our results suggest that when the knot remains in the unfolded state, the folding landscape have a smooth and funneled shape. However, when the knotted protein has to overcome the threading process this energy landscape becomes rough.

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