Takahisa Yamato

Visualizing breathing motion of internal cavities in concert with ligand migration in myoglobin

Proteins harbor a number of cavities of relatively small volume. Although these packing defects are associated with the thermodynamic instability of the proteins, the cavities also play specific roles in controlling protein functions, e.g., ligand migration and binding. This issue has been extensively studied in a well-known protein, myoglobin (Mb). Mb reversibly binds gas ligands at the heme site buried in the protein matrix and possesses several internal cavities in which ligand molecules can reside. It is still an open question as to how a ligand finds its migration pathways between the internal cavities. Here, we report on the dynamic and sequential structural deformation of internal cavities during the ligand migration process in Mb. Our method, the continuous illumination of native carbonmonoxy Mb crystals with pulsed laser at cryogenic temperatures, has revealed that the migration of the CO molecule into each cavity induces structural changes of the amino acid residues around the cavity, which results in the expansion of the cavity with a breathing motion. The sequential motion of the ligand and the cavity suggests a self-opening mechanism of the ligand migration channel arising by induced fit, which is further supported by computational geometry analysis by the Delaunay tessellation method. This result suggests a crucial role of the breathing motion of internal cavities as a general mechanism of ligand migration in a protein matrix.

Role of protein in the primary step of the photoreaction of yellow protein

We show the unexpectedly important role of the protein environment in the primary step of the photoreaction of the yellow protein after light illumination. The driving force of the trans‐to‐cis isomerization reaction was analyzed by a computational method. The force was separated into two different components: the term due to the protein‐chromophore interaction and the intrinsic term of the chromophore itself. As a result, we found that the contribution from the interaction term was much greater than that coming from the intrinsic term. This accounts for the efficiency of the isomerization reaction in the protein environment in contrast to that in solution environments. We then analyzed the relaxation process of the chromophore on the excited‐state energy surface and compared the process in the protein environment and that in a vacuum. Based on this analysis, we found that the bond‐selectivity of the isomerization reaction also comes from the interaction between the chromophore and the protein environment. Proteins 2004.

Conformational deformation in deoxymyoglobin by hydrostatic pressure

Pressure effects on the structure of sperm whale deoxymyoglobin have been studied by the normal mode analysis (NMA) and the strain tensor analysis (STA). Inhomogeneous mechanical construction of the molecule is revealed in the microscopic detail. The pressure-induced deformations of inter-helix regions are remarkably larger than those of the other parts. On the other hand, the intra-helix compressibility is shown to be relatively small.

Ab initio MO study on potential energy surfaces for twisting around C7C8 and C4–C7 bonds of coumaric acid

Abstract We have calculated potential energy surfaces (PESs) for the twisting around C7C8 and C4–C7 bonds of anionic form of coumaric acid with and without hydrogen bonds between its phenorate part and hydroxyl groups nearby, modeling the active site of Photoactive Yellow Protein. The calculation is made by means of the state-average complete active space self-consistent field theory. We revealed that the PESs of the ground and first excited states as a function of twisting angle around C7C8 are of strongly crossing type, and the crossing tendency becomes more prominent in the presence of the hydrogen bonds. We found that the PESs of the ground and first excited states as a function of twisting angle around C4–C7 bond are also of crossing type, representing double bond character.

Mechanical Property of a TIM-Barrel Protein

The mechanical response of a TIM‐barrel protein to an applied pressure has been studied. We generated structures under an applied pressure by assuming the volume change to be a linear function of normal mode variables. By Delaunay tessellation, the space occupied by protein atoms is divided uniquely into tetrahedra, whose four vertices correspond to atomic positions. Based on the atoms that define them, the resulting Delaunay tetrahedra are classified as belonging to various secondary structures in the protein. The compressibility of various regions identified with respect to secondary structural elements in this protein is obtained from volume changes of respective regions in two structures with and without an applied pressure. We found that the β barrel region located at the core of the protein is quite soft. The interior of the β barrel, occupied by side chains of β strands, is the softest. The helix, strand, and loop segments themselves are extremely rigid, while the regions existing between these secondary structural elements are soft. These results suggest that the regions between secondary structural elements play an important role in protein dynamics. Another aspect of tetrahedra, referred to as bond distance, is introduced to account for rigidities of the tetrahedra. Bond distance is a measure of separation of the atoms of a tetrahedron in terms of number of bonds along the polypeptide chain or side chains. Tetrahedra with longer bond distances are found to be softer on average. From this behavior, we derive a simple empirical equation, which well describes the compressibilities of various regions.

A computational study on the stability of the protonated Schiff base of retinal in rhodopsin

Abstract We investigated the effect of amino acids in rhodopsin on the protonation state of the Schiff base (SB) retinal. We constructed a model system consisting of SB retinal, Glu113 (counterion), and eight residues. For this model, we considered two states of the SB retinal, namely, the protonated/deprotonated state. We then performed ab initio MO calculations at the RHF/6-31g* level. As a result, the protonated state was stabler than the deprotonated state. Interestingly, we observed an additive rule for the contribution to the stabilization energy due to each amino acid. Above all, it turned out that Ser186 and Cys187 play a significant role in the stability.

A novel method for determining the electron tunneling pathway in protein

Abstract A novel method for determining the electron tunneling pathway in protein is proposed. The pathway is constructed connecting vectors of important interatomic currents of the tunneling electron. The method is applied to some ruthenium-modified azurins. From this analysis, we found that the through-space is very often used as well as the through-bond, while the hydrogen-bond is scarcely used in the important interatomic currents.

Modulation of the Absorption Maximum of Rhodopsin by Amino Acids in the C-terminus†

Vision begins when light is absorbed by visual pigments. It is commonly believed that the absorption spectra of visual pigments are modulated by interactions between the retinal and amino acids within or near 4.5 Å of the retinal in the transmembrane (TM) segments. However, this dogma has not been rigorously tested. In this study, we show that the retinal‐opsin interactions extend well beyond the retinal binding pocket. We found that, although it is positioned outside of TM segments, the C‐terminus of the rhodopsin in the rockfish longspine thornyhead (Sebastolobus altivelis) modulates its λmax by interacting mainly with the last TM segment. Our results illustrate how amino acids in the C‐terminus are likely to interact with the retinal. We anticipate our analyses to be a starting point for viewing the spectral tuning of visual pigments as interactions between the retinal and key amino acids that are distributed throughout the entire pigment.

Molecular mechanism of long-range synergetic color tuning between multiple amino acid residues in conger rhodopsin

The synergetic effects of multiple rhodopsin mutations on color tuning need to be completely elucidated. Systematic genetic studies and spectroscopy have demonstrated an interesting example of synergetic color tuning between two amino acid residues in conger rhodopsin’s ancestral pigment (p501): — a double mutation at one nearby and one distant residue led to a significant λmax blue shift of 13 nm, whereas neither of the single mutations at these two sites led to meaningful shifts. To analyze the molecular mechanisms of this synergetic color tuning, we performed homology modeling, molecular simulations, and electronic state calculations. For the double mutant, N195A/A292S, in silico mutation analysis demonstrated conspicuous structural changes in the retinal chromophore, whereas that of the single mutant, A292S, was almost unchanged. Using statistical ensembles of QM/MM optimized structures, the excitation energy of retinal chromophore was evaluated for the three visual pigments. As a result, the λmax shift of double mutant (DM) from p501 was −8 nm, while that of single mutant (SM) from p501 was +1 nm. Molecular dynamics simulation for DM demonstrated frequent isomerization between 6-s-cis and 6-s-trans conformers. Unexpectedly, however, the two conformers exhibited almost identical excitation energy, whereas principal component analysis (PCA) identified the retinal-counterion cooperative change of BLA (bond length alternation) and retinal-counterion interaction lead to the shift.

Direct measure of functional importance visualized atom-by-atom for photoactive yellow protein: Application to photoisomerization reaction

Photoreceptor proteins serve as efficient nano‐machines for the photoenergy conversion and the photosignal transduction of living organisms. For instance, the photoactive yellow protein derived from a halophilic bacterium has the p‐coumaric acid chromophore, which undergoes an ultrafast photoisomerization reaction after light illumination. To understand the structure‐function relationship at the atomic level, we used a computational method to find functionally important atoms for the photoisomerization reaction of the photoactive yellow protein. In the present study, a “direct” measure of the functional significance was quantitatively evaluated for each atom by calculating the partial atomic driving force for the photoisomerization reaction. As a result, we revealed the reaction mechanism in which the specific role of each functionally important atom has been well characterized in a systematic manner. In addition, we observed that this mechanism is strongly conserved during the thermal fluctuation of the photoactive yellow protein. We compared the experimental data of fluorescence decay constant of several different mutants and the present analysis. As a result, we found that the reaction rate constant is decreased when a large positive driving force is missing. Proteins 2004.