My current research project is to understand how ATP energy is converted to the reverse movement of myosin VI, which is the only myosin moving toward the negative end of the polar actin filament. Myosin is a superfamily of ATP-binding enzymes responsible for many essential biological functions including muscle contraction, cell division, and vesicle transport. I have adopted a multifaceted approach integrating computational modeling, protein engineering, and single molecule techniques for this project [1]. I designed a series of myosin VI constructs with artificial lever arms fused at different locations beyond the converter domain. Computational modeling including molecular dynamics simulation was used to predict the behavior of chimeric designs and select optimal fusion locations [2]. I characterized the directionality and velocity of the chimeras using in vitro motility assays, and examined single molecule movement of these chimeric constructs using the total internal reflection microscopy. Our results demonstrated that the calmodulin-bound portion is not an integral mechanistic component of the reverse stroke. Furthermore, we showed that as few as 18 residues are enough to change the directionality of myosin.
Including the project described above, all my research projects focused on energy transduction of various ATP-binding proteins, including myosins [1,3,4], helicases [5,6,7], and F1Fo ATP synthase [8]. In all these projects I applied various computational techniques to gain insights into molecular mechanism of energy transduction. The computational techniques I used include molecular dynamics simulation [9], normal mode analysis [7], reaction-diffusion equations [8], Monte Carlo simulation [3], kinetic modeling [3,5,6], and bioinformatics [4] I have also developed novel computational tools/packages freely available to analyze single molecule dwell-time distributions [3], to solve multimeric multi-state kinetics [5], and to identify pathways of allosteric communication (AlloPathFinder) [4].
To first characterize the free energy landscape of ATP, I have calculated its conformational equilibria in solution using molecular dynamics augmented with umbrella sampling methods [9]. Comparing the free energy function with several crystallographic structures of ATP analogs, we were able to calculate the energy stored in the proteins when ATP binds.
Several helicases have been studied in detail. They include T7 helicase [5], Rho transcription terminator [6], and Hepatitis C virus NS3 helicase [7]. The bacteriophage T7 helicase is a ring-shaped hexameric helicase that unwinds double-stranded DNA during DNA replication and recombination. Combining the computer simulation with biochemical data from our collaborator, we have clarified two long-standing questions: all six subunits of the helicase are catalytic, and the order of moving DNA by these subunits is sequential instead of random [5]. A recent crystal structure of another ring helicase (E1) co-crystallized with DNA supported our finding of sequential mechanism [10].
Hepatitis C virus NS3 helicase is a key component of RNA replication and has been a drug target for treating Hepatitis C [11]. Our model illustrated how ATP binding drives DNA binding regulation and DNA translocation [7], which has been illusive. Furthermore we identified critical residues participating in its allosteric interactions.
I have also studied several myosins, including myosin I [4], myosin II [4], myosin V [3,4], and myosin VI [1]. Myosin V transports intracellular cargos by walking on an actin filament step-by-step. It is beyond the time resolution to directly observe when the power stroke occurs in myosin V's chemical cycle due to several fast transition steps. I have applied my computational package for dwell-time distributions to analyze the data of myosin V. By testing different coupling models between chemistry and mechanics, we have concluded that the mechanical power stroke of myosin V corresponds to two conformations in its ADP-bound state [3].
We have also applied our program AlloPathFinder to predict pathways of conserved residues connecting the myosin ATP binding site to the lever arm [4]. We examined different conformations of myosins I, II, and V and found that most of the pathways traverse through the relay helix leading to the beginning of the lever arm, consistent with the understood need for allosteric communication in this conformation.
In summary, I have applied multiple computational techniques to gain insights into the molecular mechanism of enzymatic energy transduction for various ATP-binding proteins, and I am currently integrating the computational approach with biochemical, biophysical, and protein engineering approaches to gain mechanistic understanding of myosin VI. My future research plan is to continue the studies of ATP bioenergy transuduction using this multifaceted approach, and furthermore to design and engineer enzymes with higher efficiency to consume less ATP.
1. Liao, J. C., Bryant, Z., Elting, M. W., Delp, S. L. & Spudich, J. A. (2007). Probing the reverse directionality of myosin VI. in preparation.
2. Liao, J. C., Bryant, Z., Delp, S. L. & Spudich, J. A. (2007). Computer-aided engineering of molecular motors to move toward opposite directions. Biomedical Engineering Society 2007 Annual Meeting, Los Angeles, California.
3. Liao, J. C., Spudich, J. A., Parker, D. & Delp, S. L. (2007). Extending the absorbing boundary method to fit dwell-time distributions of molecular motors with complex kinetic pathways. Proc Natl Acad Sci U S A 104, 3171-6.
4. Tang, S.*, Liao, J. C.*, Dunn, A. R., Altman, R. B., Spudich, J. A. & Schmidt, J. P. (2007). Predicting Allosteric Communication in Myosin via a Pathway of Conserved Residues. J Mol Biol. *equal authorship
5. Liao, J. C.*, Jeong, Y. J.*, Kim, D. E., Patel, S. S. & Oster, G. (2005). Mechanochemistry of T7 DNA helicase. J Mol Biol 350, 452-75. *equal authorship
6. Adelman, J. L., Jeong, Y. J., Liao, J. C., Patel, G., Kim, D. E., Oster, G. & Patel, S. S. (2006). Mechanochemistry of transcription termination factor Rho. Mol Cell 22, 611-21.
7. Zheng, W.*, Liao, J. C.*, Brooks, B. R. & Doniach, S. (2007). Toward the mechanism of dynamical couplings and translocation in hepatitis C virus NS3 helicase using elastic network model. Proteins 67, 886-96. *equal authorship
8. Xing, J., Liao, J. C. & Oster, G. (2005). Making ATP. Proc Natl Acad Sci U S A 102, 16539-46.
9. Liao, J. C., Sun, S., Chandler, D. & Oster, G. (2004). The conformational states of Mg.ATP in water. Eur Biophys J 33, 29-37.
10. Enemark, E. J. & Joshua-Tor, L. (2006). Mechanism of DNA translocation in a replicative hexameric helicase. Nature 442, 270-5.
11. Frick, D. N. (2003). Helicases as antiviral drug targets. Drug News Perspect 16, 355-62.