When biological phenomena are viewed by classifying them into hierarchical levels, we understand that high-level biological reactions such as metabolism, growth, and learning occur by interconnection, composition, and interaction of lower-level chemical reactions of biomolecules. Therefore, I believe that ultimate understanding of higher-level biological reactions cannot be achieved without exploring the reaction mechanisms of biomolecules. I am observing the dynamics of biomolecules in real time, for instance, changes in chemical condition and structure, to disclose chemical and structural information of reaction intermediates and molecules under transition.
The majority of experiments for understanding biological phenomena have involved observing and comparing molecules in stable states before and after a chemical reaction. However, in order to chemically explain biological phenomena, it is insufficient to observe biomolecules at a stable state only. We need to observe them in unstable transitional states such as during a transition and in the middle of a reaction and clarify which chemical reaction occurs at which time.
Proteins play the main role in biochemical reactions. While undergoing dynamic changes of its three-dimensional structure, a protein manifests diverse functions such as transmitting information and catalyzing decomposition or synthesis of a substrate. There are various methods for observing a transitional state stopped in the middle of a reaction. However, stopping a reaction itself constitutes a special condition, and it is difficult to judge whether the state in the middle of the reaction is actually significant or not. It is also impossible to experimentally trap a transitional state.
Structural dynamics of proteins have a time domain ranging widely from picoseconds to seconds and even to hours. In particular, structural changes involved in a biological function and three-dimensional structure formation occur rather quickly on the order of microseconds to milliseconds. In order to observe changes that take place in such a short time, I have developed original micro flow channel devices such as a mixer and flow cell that have channels with diameters on the order of micrometers. The devices enable real-time observation of a microsecond-order reaction which has been impossible thus far.
On the micro flow channel devices which I developed, two or more kinds of liquids flow from separate channels and meet at a certain point, creating rapid changes in the solvent conditions by fluids mixing by molecular diffusion or eddy diffusion. For example, when a protein whose three-dimensional structure has been destroyed by a denaturing agent is reacted in a micro flow channel mixer with a buffer solution that restores the solvent condition to physiological conditions, reconstruction of the three-dimensional structure starts at the point where the solutions are mixed (point of reaction initiation), and the mixture of the solutions flows through the observation channel as the reaction progresses. By making the reacting mixture flow through the observation channel at a constant speed, the distance from the point of reaction initiation becomes proportional to reaction time. Therefore, we can see various stages of a reaction process by changing the point of observation along the channel. Actually, when a protein was observed by using X-ray small angle scattering and infrared spectroscopy, we successfully obtained an experimental result where the protein initially contracted into a compact shape and then slowly formed secondary structures such as α-helix and β-sheet.
The micro flow channel devices are highly compatible with measurement methods that use light. By using fluorescence lifetime spectrometry that uses a pulsed laser, we can measure the distances between amino acids and their distribution and acquire information on the protein structure such as whether the protein is rigid or flexible. With infrared absorption spectroscopy or visible absorption spectroscopy, it is possible to know the chemical states of the functional group, the active center, and the substrate that are related to a reaction. Protein information such as size and shape is available by using high-intensity X-rays available in large facilities such as SPring-8 and SACLA. Micro flow channel devices combined with micrometric use of these measurement methods has led to fine time resolution, reduced specimen consumption, and enabled observation of protein reactions previously unachievable.
This time resolution measurement system permits us to observe reactions that occur within the time range of microseconds and reactions of low-yield proteins such as membrane proteins for which it is difficult to prepare specimens. It can also be applied to the analysis of irreversible reactions caused by interactions between proteins or between a protein and substrate. Therefore, the system should be a powerful tool for deepening our understanding of the chemistry of molecular mechanisms that have yet to be discovered in molecular biology and biochemistry. With cellular transmembrane transport as my research target, I am endeavoring to elucidate the molecular mechanisms involved in highly controlled functional expression via association and dissociation of two or more biomolecules, including membrane proteins, by making use of the technologies I have developed. Based on the studies, I aim at chemical elucidation of the construction and operation principles of molecular systems consisting of conjugation of chemical reactions, interaction between proteins, and interrelation between structural changes.