Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Saturday Speaker Abstracts

FOLDING FREE ENERGY BARRIERS OF TOPOLOGICALLY KNOTTED PROTEINS Mauricio A Baez 1 ; 1 Universidad de Chile, Bioquímica y Biología Molecular, Santiago, Chile Knots in proteins are present in about 1% of all structures known, begging the question as to the importance of the knot in their particular folding mechanism and its possible functional significance. Yet, there is a lack of elementary knowledge about spontaneous knot formation in a polypeptide chain—an event that can potentially impair its folding—and about the effect of a knot on the stability of the folded state and on the kinetic barrier that separates it from the unfolded state. Moreover, at molecular level, the folding mechanism of knotted proteins remain controversial as the formation of knotted proteins has been explored by in silico simulations using coarse-grained force fields that do not consider specific contacts proposed to be key for knotting. To answer these questions, we have used optical tweezers to mechanically manipulate and untie the knotted protein from different pulling directions. This approach allowed us to quantitative characterize the folding mechanism of natural and artificial knotted proteins, and extract the free energy to form a knot by chance in the denatured state and the free energy barrier associated with the formation of a knot during the folding of the protein. We found that threading a polypeptide chain increase the folding barrier in several kcal/mol. This increment is comparable in magnitude with the energy cost to form a knot in the denatured state supporting that most of the entropic cost to form a knot is not compensated during the folding reaction. These results support that a knot is an unavoidable topological constrain, whose energy cost is transferred to the folding barrier.

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