Research Interests


Overview
My research interests have been centered on the role of conformational dynamics of biological molecules on their functions. Main information for understanding the mechanism of biological molecules is the atomic structures obtained from X-ray crystallography. From the structure, we can learn what elements are necessary for the functions. However, the structure is a static picture and it cannot explain how the biological molecules perform their functions. If we could see and observe how the molecules are moving, how it would be very helpful to understand its mechanism. This is why we use computer simulations. Using computers, we can simulate how the molecules are actually moving. However, even with the most powerful computer, it is difficult to simulate the entire event of the functions. Moreover, the biological molecules are not working in deterministic way as our macro machines do. Since the biological molecules are so small, they are working in the thermal fluctuations, i.e. under (or using) the noise from solvent or other biological molecules, we need to describe the biological event in statistical manner. In my researches, I aim to develop theoretical frameworks to describe the biological functions in terms of statistical physics, and use computational approach to simulate the real biological systems.
 
Effect of crystal packing on protein dynamics and structures
ImageX-ray crystallography provides not only structures of biological molecules, but also information on their dynamics as represented by temperature factors assigned to each atom. A high temperature factor suggests either disorder or thermal motion. Disorder means that the atom occupied different positions in different molecules in the crystal, while thermal motion refers to vibration of an atom about its average position.

Temperature factors have been used to benchmark the accuracy of computational models such as molecular dynamics simulation and normal mode analysis. However, the temperature factors include the effects of crystal packing, which are ignored in the standard simulations.

We proposed a method for normal mode analysis to include effects of crystal neighbors on the overall dynamics of the system. With neighbors, temperature factors are reproduced more accurately. One main reason of improvement is that unrealistic flexibility is suppressed by crystal packing (which also indicates that those flexible regions could be indeed very flexible in solution). Contacts between flexible loops may be important for crystallization process. Another consequence is that the external rigid body motions of the protein are coupled with the internal vibrations of the protein. We found that one third of the B-factor is from rigid body motions.
 
Energy Landscape of Protein Conformational Change

ImageProteins undergo large conformational change upon ligand binding and the conformational changes play an important role for the enzyme activity. Under the collaboration with Peter G. Wolynes, we developed a theoretical model to describe the energy landscape of protein conformational changes. In this model, in analogy to the Marcus theory of electron transfer reaction, we consider two energy surfaces correspond to different states, such as ligand bound and unbound states. Energy surfaces are extrapolated from each of steady states (energy minimum) using the normal mode. Resulting energy surface shows very high-energy barrier. From these observations we proposed the cracking model, i.e., biological molecules partially unfold in the middle of conformational change process and refold again (PNAS2003,JPC2004).

Miyashita, O., Onuchic, J. N., and Wolynes, P. G. (2003) Nonlinear elasticity, proteinquakes, and the energy landscapes of functional transitions in proteins, Proc Natl Acad Sci U S A 100, 12570-12575.
Miyashita, O., Wolynes, P. G., Onuchic, J. N. (2004) Simple energy landscape model for the kinetics of functional transitions in proteins, J. Phys. Chem. B, in press
 
Electron Transfer Reactions
ImageElectron transfer reactions are most basic chemical reaction performed by biological molecules, and moreover, they play fundamental role in energetics of biomolecules, such as photosynthesis and respiratory chains. During Ph.D. course, under the supervision of Nobuhiro Go, throughout studies on the electron transfer reaction in the cytochrome c, I developed a theoretical framework to understand the pressure dependence of biological electron transfer reactions (JPC1999), and to estimate contribution from protein conformational dynamics to the reorganization energy (JPC2000).

At UCSD, under the collaboration with Melvin Y. Okamura, I have been studying the inter-protein electron transfer reaction from cytochrome c2 to the photosynthetic reaction center. It has been shown that water molecules may play some role in the inter-protein electron transfer reaction (JPC2003).

Miyashita, O., and Go, N. (1999) Pressure dependence of protein electron transfer reactions: Theory and simulation, J. Phys. Chem. B 103, 562-571.
Miyashita, O., and Go, N. (2000) Reorganization energy of protein electron transfer reaction: Study with structural and frequency signature, J. Phys. Chem. B 104, 7516-7521.
Miyashita, O., Okamura, M. Y., and Onuchic, J. N. (2003) Theoretical understanding of the interprotein electron transfer between cytochrome c2 and the photosynthetic reaction center, J. Phys. Chem. B 107, 1230-1241.
 
Protein Binding

ImageFrom the studies on the inter-protein electron transfer reaction from cytochrome to the reaction center, it is clear that we need to full understand the binding of two proteins to understand the entire reaction event. Two molecules need to be bound correctly to make electron transfer efficiently. By combining the theoretical computation and experimental data, we constructed an atomic models of the transition state ensemble and the encounter complex (Biochemistry 2003, PNAS 2004). The obtained models can be used to examine a possibility of electron transfer reactions from loosely bound complex (2005)

Miyashita, O., Onuchic, J. N., and Okamura, M. Y. (2003) Continuum electrostatic model for the binding of cytochrome c(2) to the photosynthetic reaction center from Rhodobacter sphaeroides, Biochemistry 42, 11651-11660.
Miyashita, O., Onuchic, J. N., and Okamura, M. Y. (2004) Transition State and Encounter Complex for Fast Association of Cytochrome c2 with Bacterial Reaction Center, Proc. Natl. Acad. Sci. USA 101, 16174-16179.
Miyashita, O., Okamura, M. Y., Onuchic, J. N. (2005) Inter-protein electron transfer reactions from cytochrome c2 to photosynthetic reaction center: Electron tunneling across an aqueous interface, Proc. Natl. Acad. Sci. USA in press
 
Vibrational Energy Relaxation in Protein Molecules

ImageWe performed molecular dynamics simulation of vibrational energy transfer in a protein molecule. Dynamics of protein can be described as a set of harmonic oscillator using normal mode analysis, however, there are couplings between the normal modes due to the anharmonicity of the energy surface. At low temperature, such anharmonicity is rather weak, and the energy transfer occurs to follow a certain rule of relation between frequencies (PRL2000). However, at higher temperature, the anharmonicity is so large that such rule does not have strong effect, and yet the overlaps between the normal modes in the molecule control the energy flow (JPC2003)(with Kei Moritsugu and Akinori Kidera).

Moritsugu, K., Miyashita, O., and Kidera, A. (2000) Vibrational energy transfer in a protein molecule, Phys. Rev. Lett. 85, 3970-3973.
Moritsugu, K., Miyashita, O., and Kidera, A. (2003) Temperature dependence of vibrational energy transfer in a protein molecule, J. Phys. Chem. B 107, 3309-3317.
 
Volume Fluctuation of Protein

From molecular dynamics simulation, we calculated the volume fluctuation of human lysozyme Spectral analysis shows that low-frequency dynamics dominate the total volume fluctuation. The same aspect is found in the study using principal component analysis. This low-frequency region is related to large and slow motions of proteins. Therefore a long time dynamics simulation is necessary to describe the volume fluctuations of proteins  (with Florence Tama, Akio Kitao, Nobuhiro Go).

Tama, F., Miyashita, O., Kitao, A., and Go, N. (2000) Molecular dynamics simulation shows large volume fluctuations of proteins, Eur. Biophys. J. Biophys. Lett. 29, 472-480.