Kinetic and thermodynamic analysis of knotted proteins folding mechanism using chaotropic agents and single molecule studies: Role of non native contacts during the threading mechanism of MJ0366 from Methanocaldococcus jannaschi
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Tesis DoctoradoAuthor
Rivera, MairaAbstract
Knotted proteins constitute a group of topologically complex proteins whose backbone chain entangles in the folded state. During folding, these proteins has to thread one end of the polypeptide chain through a knotting loop in order to reach the native state. It has been suggested that the threading of the polypeptide chain is the limiting step during the folding reaction and is favored by the formation of non-native contacts. This has not been e...
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Knotted proteins constitute a group of topologically complex proteins whose backbone chain entangles in the folded state. During folding, these proteins has to thread one end of the polypeptide chain through a knotting loop in order to reach the native state. It has been suggested that the threading of the polypeptide chain is the limiting step during the folding reaction and is favored by the formation of non-native contacts. This has not been experimentally proven due to the fact that knotted proteins remain knotted when they are exposed to high concentrations of chemical denaturants. Therefore, it is difficult to study the threading in vitro. To address these problems, it has been studied the folding mechanism of MJ0366 protein from Methanocaldococcus jannaschii. We studied the folding mechanism of MJ0366 by using classical chemical denaturation studies and single molecule approaches.
MJ0366 was observed as a dimer in solution whose folding mechanism studied by chemical denaturation showed to be a four-state mechanism. In equilibrium unfolding experiments the native dimer (N2) dissociates at low GdnHCl concentrations without changes in the secondary structure, where the native monomer (M) follow a cooperative single unfolding transition stabilized by 9 kcal/mol. However, during the kinetic experiments a burst-phase intermediate (I) showed up. This intermediate is formed during the dead time of the stopped flow experiment which is 7.7 ms.
Nonetheless, from chemical denaturation experiments is impossible to determine if the polypeptide chain is fully unknotted in the unfolded state. Consequently, in order to control the topology of the unfolded state the optical tweezer experimental setup was used. Specifically, four pulling geometries were designed to mechanically unfold the monomer of MJ0366 to either tight the knot upon unfolding (KN mutant) or to untie it (UKs mutants). When the knot was tightened in force-ramp experiments, single unfolding and refolding transitions were observed, accompanied by a change in Lc consistent with the expected theoretical value. Using the Crooks fluctuation theorem, a 13 kcal/mol free energy difference was calculated from the unfolding and refolding work distributions. Moreover, at constant-force experiments the protein oscillates between two states, whose frequency of interconversion showed a linear dependence with force. From these results a refolding constant in the order of 107 s-1 was calculated. These observations suggest a two-state folding mechanism when the trefoil knot remains in the polypeptide chain whose refolding is extremely fast.
To untie the knot, three different pulling geometries were designed to control which polypeptide end thread the polypeptide chain. When the C-terminal was free to thread (UK-C and N-UK-C mutants), single unfolding transitions with a Lc consistent for the fully unfolded/unknotted proteins were observed. This suggest that both mutants can properly refold at low forces. As refolding was not observed during the relaxing cycle for UK-C and N-UK-C mutants the refolding probability, as a chance to observe an unfolding event, was used to obtain the refolding kinetics. The refolding constant for the UK-C mutant was an order of magnitude of 10-1 s-1. This is remarkably slower than that observed with the KN mutant, suggesting that the threading of the polypeptide chain increases the energy barrier of folding in MJ0366. Using the rate constants at zero force, a free energy difference of 3 kcal/mol was calculated. Since the unfolding rate constants for KN and UK-C and the stability calculated by chemical denaturation were similar, the main difference is regarded to the refolding energy barrier. In consequence, the unfolded state is 10 kcal/mol destabilized when the knot remains in this state. On the other hand, when the N-terminal was free to thread (N-UK mutant), it folds into a misfolded state since unfolding and refolding transitions present a Lc ~46% shorter than the expected. These results indicate that during the folding of MJ0366, the threading of the polypeptide chain occurs by C-terminal and this process is the limiting step of the reaction with an energy cost of 10 kcal/mol.
Chemical denaturation experiments were performed to study the stability of the KN and UKs mutants showing a free energy difference of 6 kcal/mol for the four mutants. This value is similar to that obtained when the knot is untied in the unfolded state with the UK-C mutant. Thus, is suggested that upon chemical denaturation the knot of MJ0366 is untied.
Finally, it has been studied the relevance of specific non-native contacts during the threading of the polypeptide chain by explicit solvent molecular dynamics. The non-native contacts proposed to form are R91-E34 and R45-E34, therefore E34Q, R45A and R91A mutants were performed and studied by chemical denaturation. It was observed that these mutations did not affect the refolding constant. Hence, this suggest that at least these specific non-native interactions are not relevant for the formation of the transition state of the limiting step during folding of MJ0366, ergo, are not relevant for the threading of the polypeptide chain.
In summary, from this thesis it was obtained for the first time a full kinetic and thermodynamic description for a natural knotted protein. From this information it was determined that the energy cost of threading the polypeptide chain in the unfolded state is 10 kcal/mol. This value, which is similar to the entropic cost of thread the backbone in the unfolded state, is explained almost completely by an increase in the refolding energy barrier when the backbone requires the threading to reach the energy minimum. Therefore, the threading of the polypeptide chain is the limiting step during the folding of knotted proteins and that non-native contacts are not necessary for MJ0366 folding.
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Date de publicación
2018Academic guide
Valenzuela, María Antonieta
Baez, Mauricio
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