Abstract
Knots are remarkable topological features in nature. The presence of knots in crystallographic structures of proteins have stimulated considerable research to determine the kinetic and thermodynamic consequences of threading a polypeptide chain. By mechanically manipulating MJ0366, a small single domain protein harboring a shallow trefoil knot, we allow the protein to refold from either the knotted or the unknotted denatured state to characterize the free energy profile associated to both folding pathways. By comparing the stability of the native state with reference to the knotted and unknotted denatured state we find that knotting the polypeptide chain of MJ0366 increase the folding energy barrier in a magnitude close to the energy cost of forming a knot randomly in the denatured state. These results support that a protein knot can be formed during a single cooperative step of folding but occurs at the expenses of a large increment on the free energy barrier.
Highlights
Knots are remarkable topological features in nature
Molecular dynamic simulations performed with native-centric potentials describe that the threading in MJ0366 occurs from the C-terminus via a twisted loop that is stabilized by native interactions[9,12]
This experimental strategy allowed us to compared the free energy profiles of a protein construct designed to preserve the knot in the unfolded state (Fig. 1D, construct F6C/G89C) with protein constructs designed to untie the knot in the denatured state
Summary
Knots are remarkable topological features in nature. The presence of knots in crystallographic structures of proteins have stimulated considerable research to determine the kinetic and thermodynamic consequences of threading a polypeptide chain. Besides the intramolecular non-native interactions evolved to overcome the free energy barrier of knotted proteins, it has been proposed that the cellular machinery, like chaperonins and the ribosome, can assist the folding of knotted proteins in vivo by promoting the formation of a knot in confined spaces[23,25,26], by stabilizing key intermediates and establishing new folding routes[10,26,27,28], or by modulating the collapse by hydrophobic interactions[29] These results support that knotted proteins must overcome a topological energy barrier derived from the threading of the polypeptide chain. These observations are discussed in the context mechanism of MJ0366 proposed by in silico molecular dynamic and other knotted proteins
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