Significance of Knotted Structures for Function of Proteins and Nucleic Acids - September 17-21, 2014

Significance of Knotted Structures for Function of Proteins and Nucleic Acids

Poster Session I

1 – POS Board 1 The Energetic Cost to Knot the Unfolded State of a Protein Determined by Optical Tweezers Andrés Bustamante 1 , Javiera Reyes 1 , Christian A.M. Wilson 1 , Daniel Guerra 2 .Carlos J. Bustamante 3,4,5 . Mauricio Baez 1 , 1 Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile, 2 Universidad Peruana Cayetano Heredia, Lima, Peru, 3 QB3 California Institute for Quantitative Biosciences, Berkeley, CA, USA, 4 Jason L. Choy Laboratory of Single-Molecule Biophysics, Berkeley, CA, USA, 5 Howard Hughes Medical Institute, Berkeley, CA, USA. The unfolded state of a protein is considered as a highly fluctuating state of the polypeptide chain, but its tendency to form entangled structures like knots has not been quantified. In this work, we determined the energetic cost of threading a designed knotted protein. The bacteriophage P22 ARC repressor is a homodimer, which can be converted into a single-chain monomer (mARC) adding a 15-residue glycine-rich linker to connect both subunits. Structural models show that flexible glycine-rich linker have the potential to create a knotted or not knotted chain by movements around the protein structure. Using optical tweezers, we explored the folding mechanism of mARC pulling their C and N-terminal extremes at the single molecule level. Analysis of 537 unfolding events obtained from eleven molecules of mARC show two populations of proteins characterized by contours lengths of 37 (7 molecules) and 43 nm (4 molecules). These values agree with the expected size of the polypeptide being stretching from a knotted (37 nm) or unknotted (43 nm) conformation. Furthermore, thermodynamic analysis obtained from the knotted or unknotted conformation of mARC indicates that the unfolded knotted conformation is destabilized by 6 kcal/mol with respect to the unknotted fully unfolded conformation. This situation contrasts with respect to naturally-occurring knotted proteins where knots prevail in the unfolded state. Fondecyt 11110534.

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