Takahiro Kosugi

Crucial role of protein flexibility in formation of a stable reaction transition state in an α-amylase catalysis.

Conformational flexibility of proteins provides enzymes with high catalytic activity. Although the conformational flexibility is known to be pivotal for the ligand binding and release, its role in the chemical reaction process of the reactive substrate remains unclear. We determined a transition state of an enzymatic reaction in a psychrophilic α-amylase by a hybrid molecular simulation that allows one to identify the optimal chemical state in an extensive conformational ensemble of protein. The molecular simulation uncovered that formation of the reaction transition state accompanies a large and slow movement of a loop adjacent to the catalytic site. Free energy calculations revealed that, although catalytic electrostatic potentials on the reactive moiety are formed by local and fast reorganization around the catalytic site, reorganization of the large and slow movement of the loop significantly contributes to reduction of the free energy barrier by stabilizing the local reorganization.

QM/MM Reweighting Free Energy SCF for Geometry Optimization on Extensive Free Energy Surface of Enzymatic Reaction.

We developed a quantum mechanical/molecular mechanical (QM/MM) free energy geometry optimization method by which the geometry of a quantum chemically treated (QM) molecule is optimized on a free energy surface defined with thermal distribution of the surrounding molecular environment obtained by molecular dynamics simulation with a molecular mechanics (MM) force field. The method called QM/MM reweighting free energy self-consistent field combines a mean field theory of QM/MM free energy geometry optimization developed by Yamamoto (Yamamoto, T. J. Chem. Phys.2008, 129, 244104) with a reweighting scheme for updating the MM distribution introduced by Hu et al. (Hu, H., et al. J. Chem. Phys.2008, 128, 034105) and features high computational efficiency suitable for exploring the reaction free energy surface of extensive protein conformational space. The computational efficiency with improved treatment of a long-range electrostatic (ES) interaction using the Ewald summation technique permits one to take into account global conformational relaxation of an entire protein of an enzyme in the free energy geometry optimization of its reaction center. We applied the method to an enzymatic reaction of a substrate complex of psychrophilic α-amylase from Antarctic bacterium Pseudoalteromonas haloplanktis and succeeded in geometry optimizations of the reactant and the product of the catalytic reaction that involve large conformational changes of protein loops adjacent to the reaction center on time scales reaching sub-microseconds. We found that the adjacent loops in the reactant and the product form in different conformations and produce catalytic ES potentials on the reaction center.

Theoretical study on excited states of bacteriochlorophyll a in solutions with density functional assessment.

The excited-state properties of bacteriochlorophyll (BChl) a in triethylamine, 1-propanol, and methanol are investigated with the time-dependent density functional theory by using the quantum mechanical and molecular mechanical reweighting free energy self-consistant field method. It is found that no prevalent density functionals can reproduce the experimental excited-state properties, i.e., the absorption and reorganization energies, of BChl a in the solutions. The parameter μ in the range-separated hybrid functional is therefore optimized to reproduce the differences of the absorption energies in the solutions. We examine the origin of the differences of the absorption energies in the solutions and find that sensitive balance between contributions of structural changes and solute-solvent interactions determines the differences. The accurate description of the excitation with the density functional with the adjusted parameter is therefore essential to the understanding of the excited-state properties of BChl a in proteins and also the mechanism of the photosynthetic systems.

QM/MM Geometry Optimization on Extensive Free-Energy Surfaces for Examination of Enzymatic Reactions and Design of Novel Functional Properties of Proteins

Many remarkable molecular functions of proteins use their characteristic global and slow conformational dynamics through coupling of local chemical states in reaction centers with global conformational changes of proteins. To theoretically examine the functional processes of proteins in atomic detail, a methodology of quantum mechanical/molecular mechanical (QM/MM) free-energy geometry optimization is introduced. In the methodology, a geometry optimization of a local reaction center is performed with a quantum mechanical calculation on a free-energy surface constructed with conformational samples of the surrounding protein environment obtained by a molecular dynamics simulation with a molecular mechanics force field. Geometry optimizations on extensive free-energy surfaces by a QM/MM reweighting free-energy self-consistent field method designed to be variationally consistent and computationally efficient have enabled examinations of the multiscale molecular coupling of local chemical states with global protein conformational changes in functional processes and analysis and design of protein mutants with novel functional properties.