Three-Dimensional Image Analysis of Myosin Head in Function as Captured by Quick-Freeze Deep-Etch Replica Electron Microscopy
Quick-freeze deep-etch replica electron microscopy combined with mica-flake technique provides high contrast, high time- and spatial-resolution images of protein molecules in solution, whose three-dimensional structure is well preserved. Thus, it might be quite useful to obtain structural information of individual functioning molecules, such as myosin crossbridges under in vitro motility assay conditions. With that method, we could actually show that both heads of heavy meromyosin (HMM) crossbridges are mostly straight and bound to actin filaments with about 45 degree tilt-angle under rigor conditions, whereas they attached to actin through only one head with a wide variety of angles under in vitro sliding conditions. We also demonstrated that free HMM heads are strongly kinked in the presence of ATP or ADP/inorganic vanadate (Vi) in contrast to almost straight configuration in the absence of nucleotide.
To examine more detailed structure of individual crossbridges, we tried to reconstruct the three-dimensional architecture of intramolecular subdomains of single HMM molecule. We took a series of tilted images of single HMM-ADP/Vi particle and successfully obtained its 3-D image by filtered back-projection, even with restricted range of tiltangles. By comparison of the reconstruction with the atomic model of subfragment-1 (S1) without nucleotide, we found some great structural difference, which partly might be attributable to the conformational change by nucleotide binding.
It is likely that the key of our success in 3-D reconstruction of single molecule with such resolution might be the use of quick-freeze deep-etch replica specimens. We will discuss and demonstrate the simulation results to suggest such reasoning.
Vast development of the recent research on the mechanisms of motor proteins in cell motility and muscle contraction depends largely on the introduction of three powerful entities in methodology; 1) “single-molecular physiology”1-7 in which one not only can observe the behavior but manipulate individual molecules in solution under nearly physiological conditions, 2) “structural biology”8-10 to give the precise atomic coordinates of the protein components immobilized in crystals through X-ray diffraction and analyses, and 3) “protein engineering”11-13 to provide protein materials whose amino-acids are replaced according to almost any demands from the former entities. Though the data and the interpretation of the results obtained by two former methods are complementary to each other, there is a serious and unavoidable problem that the actual data in those approaches are obtained under entirely different environments for the proteins. It is necessary to find and employ the third method to conjugate the information between them; i.e. some means to observe the ultrastructure of individual molecules and its changes under nearly physiological conditions, with reasonably high time- and spatial-resolutions. Electron microscopy could be one of few candidates suitable for such purposes, especially when coupled with quick-freezing14,15 to initially fix the materials.
KeywordsAtomic Model Heavy Meromyosin Cell BioI Unavoidable Problem Single Myosin
Unable to display preview. Download preview PDF.
- 15.Katayama, E. in Current Methods in Muscle Physiology: Advantages, Problems and Limitations (ed. Sugi, H.) in press. (Oxford Univ. Press, Oxford)Google Scholar
- 17.Katayama, E. J. Muscle Res. Cell Motil. 12, 313 (1991)Google Scholar
- 18.Katayama, E. J. Muscle Res. Cell Motil. 14, 364 (1993)Google Scholar
- 22.Katayama, E. submitted for publication.Google Scholar
- 27.Wakabayashi, K., Tokunaga, M., Kohno, I., Sugimoto, Y., Hamanaka, T., Takezawa, Y., Wakabayashi, T. & Amemiya, Y. Science (Wash. D.C.), 258, 443–447 (1992)Google Scholar
- 29.Baba, N., Satoh, H. & Nakamura, S. Bioimages, 1, 105–113 (1993)Google Scholar
- 30.Radermacher, M. in Image Analysis in Biology (Hader, D.-P. ed.) 219–249 (CRC Press Inc., New York, 1992)Google Scholar
- 31.Carazo, J.M. in Electron Tomography (Frank, J. ed.) 117–164 (Plenum Press, New York, 1992)Google Scholar