Biomedical subjects
Publications and source records attributed to Y C Sasaki.
We have successfully observed dynamical Brownian motions in an individual protein molecule and other biological ones in real-time with one-hundredth the atomic-scale precision (picometer-scale precision) using X-rays of the super photon ring-8 (SPring-8).
Time-resolved dynamical x-ray imaging of individual DNA molecules with picometer-scale precision is demonstrated for the first time. Diffracted x-ray tracking (DXT), a single-molecule experiment with x rays, monitors the rotating motions, rather than the translational motions, of a labeled nanocrystal. DXT can obtain information about the dynamics of single molecules through a quantitative analysis, since the signals from DXT are independent of the chemical conditions.
Elasticity of a two-dimensionally arranged myosin subfragment-1 (S1) was measured by using a surface forces apparatus. To prepare a two-dimensionally arranged S1-monolayer on a functionalized silver surface, we used genetically engineered Dictyostelium S1 molecules. A highly reactive cysteine residue was fused to the COOH-terminus using the recombinant DNA method. On the other hand, the maleimide groups was self-assembled onto a silver surface. Then the mutant S1 molecules were chemically bound to the functionalized silver surface at its COOH-terminus. This arrangement technique was necessary in order to create a stable S1-monolayer by chemical bond formation onto the silver surface. The occupied area of the single S1 on the silver surface was about 110 nm(2). In the interaction between the S1-monolayer and mica surfaces in aqueous solution, a long-range attractive force was observed. The elastic constants (stiffness and Young's modulus) of myosin S1 were evaluated from force-distance profiles in aqueous solution, using the Hertz theory. We found that the stiffness (or spring constant) and Young's modulus of S1 in the absence of nucleotide are 4.4 +/- 1.0 pN/nm and 0.71 +/- 0.16 GPa, respectively.
A fluorescent X-ray interference method can effectively measure nanometer-level conformational changes for non-crystallized molecules and proteins in aqueous conditions. The time-resolved technique can be used to obtain information about the dynamics of molecules and proteins. Instrumentation for time-resolved fluorescent X-ray interference has been designed. A typical interference-fringe pattern was observed with approximately 3 s of X-ray exposure time from K-fluorescent X-rays emitted from a Zn monoatomic layer on an Rh substrate. The primary X-ray beam was polychromed with a mirror for total external reflection of X-rays and was tuned to an energy level at which only Zn K radiation became optimally excited. The glancing angle of the primary X-ray beam was fixed at a glancing angle at which the total intensity of K-fluorescent X-rays emitted from Zn atoms corresponded to the maximum value. The fluorescent X-ray interference fringes were monitored with an imaging plate (IP) as a non-energy-dispersive two-dimensional detector. The exposed interference fringes on the IP were integrated along the direction of the fringes. The integrated fringes were in close agreement with a theoretical estimate based on the interference among transmitted and reflected waves at interfaces in the sample.
We have characterized the functional protein, myosin subfragment 1 (S1), attached to a gold substrate by the sulfhydryl groups of cysteine in proteins. The amino groups of the regulatory light chain (RLC) isolated from myosin were labeled with a radioisotope (125I), and the labeled RLC was incorporated into S1 from which the RLC had been removed. The radiation from 125I showed that S1 molecules had attached to the gold and, through the interference effect of the monochromatic radiation from 125I, provided information about the position of labeled RLC sites in the S1 monolayer. The interference fringes showed that the RLC was located close to the gold surface and that all of the adsorbed S1 molecules had the same orientation. We confirmed that the motor function of S1 on the gold surface is maintained by observing sliding movement at low ionic strength and by observing the detachment at high ionic strength of fluorescent actin filaments in the presence of ATP. We also found that the adsorbed S1 molecules were not removed from the Au surface by a reducing agent. Thus the Au-S bond is more stable than the S-S bond.