Biophysical Society Thematic Meeting - October 25-30, 2015

Polymers and Self Assembly: From Biology to Nanomaterials Poster Session II

37-POS Board 37 Mechanical Tightening and Untying of a Trefoil-Knotted Protein by Optical Tweezers Maira Rivera , Andrés Bustamante, Christian A. Wilson, Mauricio Baez. Universidad de Chile, Santiago, Chile According to the funnel hypothesis, the roughness of the energetic landscape increases along with the topological complexity of proteins and thus, the chance of produce misfolded structures. Knotted proteins are examples of topological complexity, but it is not clear how these proteins deal with the folding because experimentally is difficult to determine how the folding mechanism is coupled with the threading of the polypeptide chain. To solve this problem, we have studied the mechanical unfolding of a trefoil knotted protein (MJ0366 of Methanocaldococcus jannaschii ) using the experimental setup of optical tweezers. Two mutants were created, one to pull MJ0366 from the ends of the polypeptide chain to tight the knot, and other mutant in order to untie the protein by pulling from key residues of the structure. When the knot was tightened, MJ0366 showed a two state folding mechanism characterized by the presence of a single unfolding and refolding transitions with average forces of 19.8 pN and 13.6 pN respectively. The contour length was 7.8 nm shorter than expected. This could be associated in part with the presence of the knot in the structure, although the formation of trefoil knot decreases the contour length in about 5 nm. Free energy of unfolding reaction, calculated using the Crooks fluctuation theorem, is 13.6 kcal/mol around 4.4 kcal/mol higher than the obtained in experiments performed with chaotropic agents. In the case of the MJ0366 mutant whose knot is untied upon pulling, preliminary experiments show a complex folding mechanism with multiple transitions of unfolding and refolding. These results suggest that threading of the polypeptide chain creates a rougher landscape for folding, but once the knot is formed, the folding landscape becomes smooth. Funded by Fondecyt: 1151274, Anillo ACT- 1107, CONICYT fellowship N°21130254.

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