SINGLE MOLECULE NANO-BIOSCIENCE

Toshio Yanagida

Graduate School of Frontier Bioscience and Graduate School of Medicine, Osaka University, 1-3, Yamadaoka, Suita, Osaka, 565-0871, Japan

1. Introduction

Biomolecules assemble to form molecular machines such as molecular motors, cell signal processors, DNA transcription processors and protein synthesizers to fulfill their functions. Their collaboration allows the activity of biological systems. The reactions and behaviors of molecular machines vary flexibly while responding to their surroundings. This flexibility is essential for biological organisms. The underlying mechanism of molecular machines is not as simple as that expected from analogy with man-made machines. Since molecular machines are only nanometers in size and has a flexible structure, it is very prone to thermal agitation. Furthermore, the input energy level is not much difference from average thermal energy, kBT. Molecular machines can thus operate under the strong influence of this thermal noise, with a high efficiency of energy conversion. They would not overcome thermal noise but effectively use it for their functions. This is in sharp contrast to man-made machines that operate at energies much higher than the thermal noise. In recent years, the single molecule detection (SMD) and nano-technologies have rapidly been expanding to include a wide range of life science. The dynamic properties of biomolecules and the unique operations of molecular machines, which were previously hidden in averaged ensemble measurements, have now been unveiled. The aim of our research is to approach the engineering principle of adaptive biological system by uncovering the unique operation of biological molecular machines. I survey our SMD experiments designed to investigate molecular motors, enzyme reactions, protein dynamics and cell signaling, and discuss the mechanism of biological molecular machines.

2. Single-molecule detection (SMD) techniques

How advantageous is SMD for investigations? Observing and manipulating biomolecules allows their dynamic behaviors to be directly revealed, as has been demonstrated for motor proteins. Reactions of biological molecules are generally stochastic. Therefore, even if the reactions of molecules are initiated at the same time, they cannot be synchronized, so the dynamic behaviors of individual molecules are averaged and hidden in ensemble-averaged measurements. In vivo, biomolecules work in dynamic and complicated heterogeneous systems, involving different kinds of molecules such as cell-signaling proteins. It is difficult to quantitatively detect dynamic behaviors of target molecules in such systems by ensemble-averaged measurements. The SMD techniques are expected to overcome these difficulties and have already been successfully applied to study the dynamic properties of biological molecules such as motor proteins, enzymes, RNA polymerase and cell-signaling proteins.

The SMD techniques are based on two key technologies for single-molecule imaging and single-molecule nanomanipulation. First, the imaging technique will be explained. The size of biomolecules and even their assemblies are in the order of nanometers, so they are too small to observe by optical microscopy. To overcome this problem, biomolecules can be fluorescently labeled and visualized using fluorescence microscopy. Single fluorophores in aqueous solution were first observed in 1995 by using total internal reflection fluorescence microscopy (TIRFM) and conventional inverted fluorescence microscopy. The major problem to overcome when visualizing single fluorophores in aqueous solution is the huge background noise, which can be caused by Raman scattering from water molecules, incident light breaking through filters, luminescence arising from the objective lens, immersion oil and dust. In this system, the evanescent field was formed when the laser beam was totally reflected by the interface between the solution and the glass. The evanescent field was not restricted to the diffraction limit of light, thus it could be localized close to the glass surface, which resulted in the penetration depth (~150 nm) being several-fold shorter than the wavelength of light. Therefore, the illumination was restricted to fluorophores either bound to the glass surface or located close by, thereby reducing the background light. Furthermore, by careful selection of optical elements, the background noise could be reduced by 2000-fold compared with that of conventional fluorescence microscopy. This made it possible to clearly observe single fluorophores in aqueous solution. Fluorescence measurements from fluorophores attached to biomolecules and ligands allow the detection of, for example, the movements, conformational changes, enzymatic reactions and cell-signal processes of biomolecules at the single molecule level.

The second key technology is single-molecule nanomanipulation. Biomolecules and even single molecules can be captured by a glass needle or by beads trapped by optical tweezers. The optical tweezers are the tool to trap and manipulate particles of 25 nm to 25 µm in diameter by the force of laser radiation pressure. The particle is trapped near the focus of laser light when focused by a microscope objective with a high numerical aperture. The optical tweezers produce forces in the piconewton range on the particles. Biomolecules are too small to be directly trapped by the optical tweezers, so they are generally attached to an optically -trapped bead. Microneedles or a bead trapped by a laser act as a spring that expands in proportion to the applied force. Thus, the force and the displacement caused by the biomolecules can be measured. The displacement of a microneedle and a bead has been determined with a subnanometer accuracy, much less than the diffraction limit of an optical measurement. This accuracy of displacement corresponds to the sub-piconewton accuracy in the force measurements. Thus, the mechanical property of biomolecules can be determined directly at the single-molecule level. Furthermore, combined with the single-molecule imaging technique, simultaneous measurements of mechanical and chemical reactions of single biomolecules are possible.

3. Movement and ATPase turnovers of biological molecular motors

The SDM techniques were first used to...