Structural Analysis of Dymamics of Celluar Motors

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Based on crystal structures of the motor domain of Dictyostelium myosin II, we carried out of the structure-function analysis of this motor domain. We constructed many types of Dictyostelium myosin II mutants, which were trapped at a specific step of ATP hydrolysis cycle. Biochemical and biophysical studies on these mutants have shown how the three-dimensional structure of the motor domain rearranges at each step of ATP hydrolysis cycle, and how these changes are finally transmitted to the actin-binding site and the converter domain to generate sliding and force generation on an actin filament. Furthermore, we carried out the GFP-based FRET measurements of the fusion protein consisting of GFP, the motor domain and BFP. These FRET measurements have shown that the converter domain really swings at the isomerization step first and then swings back at the Pi release step. The latter step is likely to correspond to the stroke step. We have then constructed a prism-based total reflection fluorescence microscope that can visualize fluorescence spectra of many single molecules in a microscope filed at the same time at the video rate. By using this novel technique, we have detected that the converter of myosin motor domain is trapped in a particular orientation in the presence of various types of nucleotide analogs such as MgADP.Vi, MgADP.BeFx and MgADP.AlFx, consistent with the ensemble measurements mentioned above.