@article {404, title = {Energy landscape of knotted protein folding.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {109}, year = {2012}, month = {2012 Oct 30}, pages = {17783-8}, abstract = {Recent experiments have conclusively shown that proteins are able to fold from an unknotted, denatured polypeptide to the knotted, native state without the aid of chaperones. These experiments are consistent with a growing body of theoretical work showing that a funneled, minimally frustrated energy landscape is sufficient to fold small proteins with complex topologies. Here, we present a theoretical investigation of the folding of a knotted protein, 2ouf, engineered in the laboratory by a domain fusion that mimics an evolutionary pathway for knotted proteins. Unlike a previously studied knotted protein of similar length, we see reversible folding/knotting and a surprising lack of deep topological traps with a coarse-grained structure-based model. Our main interest is to investigate how evolution might further select the geometry and stiffness of the threading region of the newly fused protein. We compare the folding of the wild-type protein to several mutants. Similarly to the wild-type protein, all mutants show robust and reversible folding, and knotting coincides with the transition state ensemble. As observed experimentally, our simulations show that the knotted protein folds about ten times slower than an unknotted construct with an identical contact map. Simulated folding kinetics reflect the experimentally observed rollover in the folding limbs of chevron plots. Successful folding of the knotted protein is restricted to a narrow range of temperature as compared to the unknotted protein and fits of the kinetic folding data below folding temperature suggest slow, nondiffusive dynamics for the knotted protein.}, keywords = {Evolution, Molecular, Kinetics, Models, Molecular, Molecular Dynamics Simulation, Mutation, Protein Folding, Proteins}, issn = {1091-6490}, doi = {10.1073/pnas.1201804109}, author = {Joanna I. Sulkowska and Noel, Jeffrey K and Onuchic, Jos{\'e} N} } @article {403, title = {The unique cysteine knot regulates the pleotropic hormone leptin.}, journal = {PLoS One}, volume = {7}, year = {2012}, month = {2012}, pages = {e45654}, abstract = {Leptin plays a key role in regulating energy intake/expenditure, metabolism and hypertension. It folds into a four-helix bundle that binds to the extracellular receptor to initiate signaling. Our work on leptin revealed a hidden complexity in the formation of a previously un-described, cysteine-knotted topology in leptin. We hypothesized that this unique topology could offer new mechanisms in regulating the protein activity. A combination of in silico simulation and in vitro experiments was used to probe the role of the knotted topology introduced by the disulphide-bridge on leptin folding and function. Our results surprisingly show that the free energy landscape is conserved between knotted and unknotted protein, however the additional complexity added by the knot formation is structurally important. Native state analyses led to the discovery that the disulphide-bond plays an important role in receptor binding and thus mediate biological activity by local motions on distal receptor-binding sites, far removed from the disulphide-bridge. Thus, the disulphide-bridge appears to function as a point of tension that allows dissipation of stress at a distance in leptin.}, keywords = {Cysteine, Humans, Kinetics, Leptin, MCF-7 Cells, Models, Molecular, Oxidation-Reduction, Signal Transduction}, issn = {1932-6203}, doi = {10.1371/journal.pone.0045654}, author = {Haglund, Ellinor and Joanna I. Sulkowska and He, Zhao and Feng, Gen-Sheng and Jennings, Patricia A and Onuchic, Jos{\'e} N} } @article {409, title = {Slipknotting upon native-like loop formation in a trefoil knot protein.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {107}, year = {2010}, month = {2010 Aug 31}, pages = {15403-8}, abstract = {Protein knots and slipknots, mostly regarded as intriguing oddities, are gradually being recognized as significant structural motifs. Recent experimental results show that knotting, starting from a fully extended polypeptide, has not yet been observed. Understanding the nucleation process of folding knots is thus a natural challenge for both experimental and theoretical investigation. In this study, we employ energy landscape theory and molecular dynamics to elucidate the entire folding mechanism. The full free energy landscape of a knotted protein is mapped using an all-atom structure-based protein model. Results show that, due to the topological constraint, the protein folds through a three-state mechanism that contains (i) a precise nucleation site that creates a correctly twisted native loop (first barrier) and (ii) a rate-limiting free energy barrier that is traversed by two parallel knot-forming routes. The main route corresponds to a slipknot conformation, a collapsed configuration where the C-terminal helix adopts a hairpin-like configuration while threading, and the minor route to an entropically limited plug motion, where the extended terminus is threaded as through a needle. Knot formation is a late transition state process and results show that random (nonspecific) knots are a very rare and unstable set of configurations both at and below folding temperature. Our study shows that a native-biased landscape is sufficient to fold complex topologies and presents a folding mechanism generalizable to all known knotted protein topologies: knotting via threading a native-like loop in a preordered intermediate.}, keywords = {Algorithms, Archaea, Archaeal Proteins, Crystallization, Databases, Protein, Models, Molecular, Molecular Dynamics Simulation, Protein Conformation, Protein Folding, Protein Multimerization, Protein Structure, Secondary, Protein Structure, Tertiary, Thermodynamics}, issn = {1091-6490}, doi = {10.1073/pnas.1009522107}, author = {Noel, Jeffrey K and Joanna I. Sulkowska and Onuchic, Jos{\'e} N} }