Yoshiharu Ishii

Single Molecule Detection in Life Sciences

In recent years, the development of single molecule detection (SMD) techniques has opened up a new era of life science. The dynamic properties of biomolecules and the unique operations of molecular machines, which were hidden in averaged ensemble measurements, have been unveiled. The SMD techniques have rapidly been expanding to include a wide range of life science. The experiments on molecular motors, DNA transcription, enzyme reactions, protein dynamics, and cell signaling are summarized in this short review.

FLUORESCENCE RESONANCE ENERGY TRANSFER BETWEEN SINGLE FLUOROPHORES ATTACHED TO A COILED-COIL PROTEIN IN AQUEOUS SOLUTION

Abstract Fluorescence resonance energy transfer (FRET) is a technique to detect the structural changes in biomolecules. We extended this technique to the single-molecule level in aqueous solution, by combining it with total internal reflection fluorescence microscopy. Both multiple color images and fluorescence spectra at the single-molecular level were obtained to determine FRET from Cy3 to Cy5 attached to α-tropomyosin (αTm), a coiled-coil of homodimer. The FRET properties observed between single fluorophores were consistent with the premise of FRET. On excitation at the donor, the fluorescence of the donor decreased and the fluorescence of the acceptor increased. Photobleaching of one of the fluorophore affected the fluorescence of the other as predicted by the mechanism of FRET. Photobleaching occurred in a single step, confirming that the donor and acceptor fluorophores were single. Large FRET efficiency (78%) was obtained in agreement with a coiled-coil structure and the FRET decreased when the protein was denatured into two polypeptide chains. Thus, we demonstrated that it is possible to detect the assembly–disassembly of individual protein molecules as well as conformational changes occurring within a single protein molecule.

Spectral Fluctuation of a Single Fluorophore Conjugated to a Protein Molecule

We have measured the fluorescence spectra of a single fluorophore attached to a single protein molecule in aqueous solution using a total internal reflection fluorescence microscope. The most reactive cysteine residue of myosin subfragment-1 (S1) was labeled with tetramethylrhodamine. The spectral shift induced by a change in solvent from aqueous buffer to methanol in both single-molecule and bulk measurements were similar, indicating that, even at the single molecule level, the fluorescence spectrum is sensitive to microenvironmental changes of fluorophores. The time dependence of the fluorescence spectra of fluorophores attached to S1 molecules solely showed a fluctuation with time over a time scale of seconds. Because the fluorescence spectra of the same fluorophores directly conjugated to a glass surface remained constant, the spectral fluctuation observed for the fluorophores attached to S1 is most likely due to slow spontaneous conformational changes in the S1 molecule. Thus, single-molecule fluorescence spectroscopy appears to be a powerful tool to study the dynamic behavior of single biomolecules.

Brownian motion, fluctuation and life.

The measurements of dynamic behaviors of biomolecules in relation to their functions have been allowed using single molecule measurements. Thermal Brownian motion causes random step motion of motor proteins and structural fluctuation of protein molecules between multiple states. In hierarchic structure of life, the fluctuation is modulated. Random fluctuation is biased to directional motion and reactions as a result of interaction of proteins. The fluctuation of kinetic state of signaling proteins results in polarization and localization of cells. A recognition process in brain is also explained by the equation analogous to biochemical reaction at the molecular level. Thus dynamic processes originated from thermal motion may play an important role in activation processes in life.

Single molecule nanomanipulation of biomolecules

The development of nanomanipulation techniques has given investigators the ability to manipulate single biomolecules and to record mechanical events of biomolecules at the single molecule level. The techniques were developed to elucidate the mechanism of molecular motors. We can directly monitor the unitary process of the mechanical work and the energy conversion processes by combining these techniques with the single molecule imaging techniques. Our results strongly suggest that the sliding movement of the actomyosin motor is driven by Brownian movement. Other groups have reported data that are more consistent with the lever arm model. These methods and imaging techniques enable us to monitor the behavior of biomolecules at work and will be applied to other molecular machines.

IMAGING AND NANO-MANIPULATION OF SINGLE BIOMOLECULES

We have developed a new technique for imaging single fluorescent dye molecules by refining epifluorescence and total internal reflection fluorescence microscopies. In contrast to previously reported single fluorescent molecule imaging methods, in which specimens were immobilized on an air-dried surface, our method enables video-rate imaging of single molecules in aqueous solution. This approach enabled us to directly image the processive movement of individual fluorescently labeled kinesin molecules along a microtubule. This method was also used to visualize individual ATP turnover reactions of single myosin molecules. The method can be combined with molecular manipulation using an optical trap. A single kinesin molecule attached to a polystyrene bead was brought into contact with a microtubule adsorbed onto the glass surface. The lifetime of bound Cy3-nucleotide in the absence or presence of the microtubule was 10 s or 0.08 s, respectively, showing that ATPase activity of the kinesin is strongly activated by microtubules. As the present system is equipped with a nanometer sensor, elemental steps of a single kinesin molecule can also be measured. By simultaneously measuring the individual ATP turnovers and elementary mechanical events of a single kinesin molecule, we will be able to obtain a clear answer to the fundamental problem of how the mechanical events are coupled to the ATPase reaction.

Single molecule dynamics in life science

A Road Map to Single Molecule Dynamics Single Molecule Study for Elucidating the Mechanism used by Biosystems to Utilize Thermal Fluctuations Imaging and Molecular Motors Ion Channel Signal Transduction across the Plasma Membrane Dynamics of Membrane Receptors: Single Molecule Tracking of Quantum Dot Liganded Epidermal Growth Factor Studying Dynamics of Ligand-Receptor Complexes by Single-Molecule Techniques RNA in Cells Protein Dynamics and Interactions Two Rotary Motors of ATP Synthase Single Molecule FRET Studies of Helicases and Holliday Junctions High-speed Atomic Force Microscopy for Nano-visualization of Biomolecular Processes Force-clamp Spectroscopy of Single Proteins

Spectral heterogeneity in single light-harvesting chlorosomes from green sulfur photosynthetic bacterium chlorobium tepidum.

Abstract The fluorescence emission properties of single chlorosomes from the green sulfur photosynthetic bacterium Chlorobium (Chl.) tepidum are studied for the first time, using a total internal reflection fluorescence microscope. The fluorescence peak positions of bacteriochlorophyll (BChl)-c self-aggregates in a single chlorosome of Chl. tepidum were widely distributed in the wavelength region between 750 and 768 nm, and the standard deviation (s.d. = 4.1 nm, n = 51) was larger than that of single chlorosomes of Chloroflexus (Cfl.) (s.d. = 1.9 nm, n = 50). The spectral heterogeneity among single chlorosomes from Chl. tepidum was in sharp contrast to those from Cfl. aurantiacus. The difference of chlorosomal spectral properties between Chl. tepidum and Cfl. aurantiacus at the single-unit level would be ascribed to the homolog composition of BChl-c—chlorosomes of Chl. tepidum have BChl substituted with various alkyl groups at both the 8- and 12-positions, whereas light-harvesting BChl-c molecules in Cfl. chlorosomes have the same substituents at the 8- (ethyl group) and 12- (methyl group) positions.

Single molecule processes on the stepwise movement of ATP-driven molecular motors

Movement is a fundamental characteristic of all living things. This biogenic function that is attributed to the molecular motors such as kinesin, dynein and myosin. Molecular motors generate forces by using chemical energy derived from the hydrolysis reaction of ATP molecules. Despite a large number of studies on this topic, the chemomechanical energy transduction mechanism is still unsolved. In this study, we have investigated the chemomechanical coupling of the ATPase cycle to the mechanical events of the molecular motor kinesin using single molecule detection (SMD) techniques. The SMD techniques allowed to detection of the movement of single kinesin molecules along a microtubule and showed that kinesin steps mainly in the forward direction, but occasionally in the backward. The stepping direction is determined by a certain load-dependent process, on which the stochastic behavior is well characterized by Feynmans thermal ratchet model. The driving force of the stepwise movement is essentially Brownian motion, but it is biased in the forward direction by using the free energy released from the hydrolysis of ATP.

Single molecule measurements and molecular motors

Single molecule imaging and manipulation are powerful tools in describing the operations of molecular machines like molecular motors. The single molecule measurements allow a dynamic behaviour of individual biomolecules to be measured. In this paper, we describe how we have developed single molecule measurements to understand the mechanism of molecular motors. The step movement of molecular motors associated with a single cycle of ATP hydrolysis has been identified. The single molecule measurements that have sensitivity to monitor thermal fluctuation have revealed that thermal Brownian motion is involved in the step movement of molecular motors. Several mechanisms have been suggested in different motors to bias random thermal motion to directional movement.