Associate Professor, Nanotechnology Research Center, Research Institute for Electronic Science, Hokkaido University
Imaging and manipulation of biological molecules in liquids by AFM
My research interests are focused on the development of an atomic force microscope (AFM), which allows us to image and manipulate biological samples under physiological conditions. In order to image their native structures, the molecules must be physically absorbed onto a surface without being fixed in liquids. Recently, we found that a frequency modulation (FM) method along with a self-oscillation technique can be utilized in liquid environments. The clear images of the assembled proteins were obtained without chemical fixations using an applied force weaker than the force used in conventional techniques. The height of the assembled proteins, observed in FM mode, was significantly higher than that observed in amplitude modulation (AM) mode in liquids as well as reported for dried samples with AFM, indicating that the FM technique is suitable for use in liquid environments. Moreover, the height was consistent with diameters obtained by electron microscopy, indicating that the proteins were only slightly compressed by the AFM tip during the scanning
By tethering single molecules between a substrate and an AFM tip, we can obtain force-extension curves, which provide information about the mechanical stability, such as the elasticity of the molecule. Using a modified force spectroscopy, we measured the viscoelasticity of protein molecules with two conformational isomers. A clear out-of-phase response against the external oscillation was observed in the case of the proteins with enzymatic activity, and in-phase response was observed for those without activity. Moreover, we determined that the dynamics of a protein drastically changed as it underwent discontinuous transition from a folded structure to a denatured one, suggesting that refolding of a partially unfolded region of the enzyme leads to the out-of-phase response. These studies show that our technique is very useful for studying the folding mechanism of proteins at the level of a single molecule.
