Hiroaki Yokota

Non‐Contact Electrostatic Surface Force Imaging of Single Protein Filaments using Intermolecular Force Microscopy

An electrostatic force image from a single protein filament was measured and visualized in pure water using Intermolecular Force Microscopy (IFM). Refining atomic force microscopy in combination with very flexible cantilevers and an optical feedback system that controlled the position of the cantilever allowed these non-contact measurements to be made with piconewton and nanometer accuracy.Cantilever probes with positively charged ZnO whisker crystals as the scanning stylus were used to probe the electrostatic features of myosin filament surface. In contrast to topographical images obtained by a conventional atomic force microscope (AFM), non-contact force images revealed two areas on the myosin filament with different charge densities. A central bare zone had the greatest negative charge in the filament, which neutralized the repulsive interaction between the charged probe and a charged glass surface. The remainder of the filament was less negatively charged, because positively charged heads decreased the net charge density of the filament. This interpretation was supported by the observation that electrostatic repulsive forces exist between the S1 self-assembled monolayer formed on gold and the positively charged whiskers in a low salt solution. Thus, it is the electrostatic features of the protein surface, rather than the surface topography, that are measured and visualized at the molecular level.

Single-Molecule Imaging of Interaction between Dextran and Glucosyltransferase from Streptococcus sobrinus

Using total internal reflection fluorescence microscopy, we directly observed the interaction between dextran and glucosyltransferase I (GTF) of Streptococcus sobrinus. Tetramethylrhodamine (TMR)-labeled GTF molecules were individually imaged as they were associating with and then dissociating from the dextran fixed on the glass surface in the evanescent field. Similarly dynamic behavior of TMR-labeled dextran molecules was also observed on the GTF-fixed surface. The duration of the stay on the surface (dwell time) was measured for each of these molecules by counting the number of video frames that had recorded the image. A histogram of dwell time for a population of several hundred molecules indicated that the GTF-dextran interaction obeyed an apparent first-order kinetics. The rate constraints estimated for TMR-labeled GTF at pH 6.8 and 25 degrees C in the absence and presence of sucrose were 9.2 and 13.3 s(-1), respectively, indicating that sucrose accelerated the dissociation of GTF from dextran. However, the accelerated rate was still much lower than the catalytic center activity of GTF (> or = 25 s(-1)) under comparable conditions.

Blinking and its Effect on Single Molecule FRET Measurements

A repeated cycle of fluorescence turning-on and -off or blinking behavior has been observed for single nanoparticles and green fluorescent protein (GFP) molecules. In this study, we found that chemical fluorescent dyes such as Cy5 and IC5 also show blinking. In single molecule FRET (smFRET) measurements using these fluorophores, the blinking effect should be taken into consideration, because the blinking of an acceptor results in apparent changes in the FRET efficiency. To distinguish the blinking from real FRET changes, the fluorescent state of an acceptor should be monitored at the same time. Donors (tetramethylrhodamine) and acceptors (IC5) were alternately excited by switching laser light between at 532 nm and 633 nm using electronic shutters. In addition, the donor and acceptor fluorescences were simultaneously imaged using a dual-view apparatus and recorded by an ICCD video camera at a video rate of 30 frames/sec. We found that some of smFRET efficiency changes were due to the blinking of the accepter. Thus, we could eliminate the blinking effect from smFRET measurements. After eliminating the blinking effect, we could analyze the FRET data from doubly labeled actin fixed on a glass surface.

DNA Unwinding Mechanism by Escherichia Coli ReCQ: Implications for DNA Replication

The RecQ protein family, a group of highly conserved DNA helicases including Escherichia coli RecQ, Saccharomyces cerevisiae Sgs1, Shizosaccharomyces Rqh1 and five gene products in humans, plays important roles in maintaining genomic stability. A biochemical analysis showed that E. coli RecQ unwinds various short DNA substrates (19 bp) with a fork containing a gap on the leading strand more efficiently than those with a gap on the lagging strand. A genetic analysis showed that the recQ deletion suppresses the induction of SOS response and the cell filamentation in cells that carry the dnaE486 (a mutation in the DNA polymerase III α-catalytic subunit), causing high proportion of anucleated cells. These previous results suggest that RecQ functions to generate an initiating signal to recruit RecA for SOS induction at stalled replication forks, which are required for the cell cycle checkpoint. On the other hand, a recent report indicates that higher concentration of RecQ is required for unwinding long DNA than for the short DNA substrates as described above, suggesting that RecQ can unwind DNA from not only the end but also the middle of the DNA. In this study, we analyzed RecQ helicase activity using linearized plasmid DNA (2∼3 kb) with and without a fork to investigate DNA unwinding mechanism of RecQ in more detail and would like to discuss how RecQ rescues stalled replication forks.