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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