44477-G6
Using Chemical Energy at a Single Molecule Level: Chemomechanical Coupling of Molecular Motors
In the past year, we focused on developing fast and efficient sampling method so that large scale protein conformational changes such as the ATP hydrolysis induced domain motions of motor proteins can be studied effectively. We made significant progress in methodology development for enhanced sampling in energy and configuration space: To efficiently enhance the sampling, we introduced the self-adaptive integrated temperature sampling (ITS) method, which is easily applied to very large systems and allows very fast thermodynamics calculations. The method was shown to be significantly more efficient than other existing methods, such as the replica exchange method. The method was applied to study the thermodynamics and kinetics of protein folding, the protein conformational changes, and polypeptide aggregation. Some of its applications are summarized below.
Results on the Folding Mechanism and Folding Rate of trpzip2
An extensive enhanced-sampling simulation for the folding of trpzip2 in the implicit solvent was performed, resulted in a trajectory of the total length of 4 microseconds, during which about 100 folding and unfolding events were observed. The results were analyzed and after re-weighting, converged free energy profiles were obtained. The free energy is calculated as a function of the root mean square displacement (RMSD) of the alpha-carbon in comparison with the NMR experimental structure and the radius of gyration, and as a function of RMSD and the number of native hydrogen bonds. By examining the free energy profiles, a mechanism for the folding of trpzip2 was obtained: During the early stage of protein folding, where RMSD decrease from about 10 to 5 A, there is a significant decrease of the radius of gyration but no obvious change of the number of native hydrogen bonds, indicating that the early stage of protein folding is mainly driven by the hydrophobic collapse of the polypeptide chain. On the other hand, at the later stage of folding, the radius of gyration remains largely constant while the number of native hydrogen bonds increases, indicating that this stage of the formation of the hairpin structure is driven by the hydrogen bond formation. The calculation of the potential of mean force shows that the hairpin formation crosses a barrier at the number of native hydrogen bond of 1, indicating that at the transition state only one hydrogen bond is formed. These results combined with the analysis of the most stable intermediates suggest that the beta-hairpin is formed by a zipping out mechanism: the most inner hydrogen bond formation is the rate limiting step and once it is formed, the other hydrogen bonds are formed sequentially.
To calculate the rate of trpzip folding, the potential of mean force was obtained as a function of the RMSD. We also calculated the diffusion constant using the NMR structure of trpzip2 with TIP3P explicit water molecules. Using Kramer’s equation we obtained a folding time of
Results on Polypeptide Aggregation
Extensive simulations were performed on the aggregation of the polypeptide with the sequence of GGVVIA. In these simulations, a variety of system sizes, which contain 10, 20 or 40 polypeptide chains, were used. Consistent with earlier simulations and the experimental observations of Eisenberg and coworkers, the polypeptides aggregate to form ordered beta-sheet structures: In each layer of the beta-strand polypeptides interact with their neighbors through hydrogen-bonding interactions and they are ordered in a parallel fashion; Between neighboring layers, polypeptides interact mainly through hydrophobic interactions between of side chains, and the neighboring layers have a very high tendency to be anti-parallel to each other. The micro-crystal like structure becomes more ordered with the increase of number of polypeptides that are present in the aggregate, presumably due to a better packing. Nevertheless, for all system sizes, aggregation was observed, indicating the high tendency of aggregation of this polypeptide sequence, which is actually believed to the sequence responsible in the Aβ amyloid. Besides crystalline structures, the simulations showed that left-handed fibrillar structures are also formed, consistent with experimental observations.
Results on the conformational change of kinesin
The enhanced sampling method was also applied to study the conformational change of kinesin as a result of substrate binding at the catalytic domain, an important process in the chemomechanical coupling mechanism of kinesin. In these simulations, the crystal structure with ADP bound at the catalytic domain was used as the initial structure and the ADP was substituted by an ATP before the simulations. Explicit water model was used. These studies showed that upon the substitution of the ADP by ATP, the protein motor takes a conformational change characterized by the re-docking of the neck-linker, consistent with experimental results. The analysis of the conformational pathway is revealing a molecular detailed pathway for the allosteric interaction between the ATP binding domain and the neck-linker, and that between the ATP binding and microtubule binding domains.
