Coordinated design of cofactor and active site structures in development of new protein catalysts

New methods for the synthesis of artificial metalloenzymes are important for the construction of novel biocatalysts and biomaterials. Recently, we reported new methodology for the synthesis of artificial metalloenzymes by reconstituting apo-myoglobin with metal complexes (Ohashi, M. et al., Angew Chem., Int. Ed. 2003, 42, 1005-1008). However, it has been difficult to improve their reactivity, since their crystal structures were not available. In this article, we report the crystal structures of M(III)(Schiff base).apo-A71GMbs (M = Cr and Mn). The structures suggest that the position of the metal complex in apo-Mb is regulated by (i) noncovalent interaction between the ligand and surrounding peptides and (ii) the ligation of the metal ion to proximal histidine (His93). In addition, it is proposed that specific interactions of Ile107 with 3- and 3'-substituent groups on the salen ligand control the location of the Schiff base ligand in the active site. On the basis of these results, we have successfully controlled the enantioselectivity in the sulfoxidation of thioanisole by changing the size of substituents at the 3 and 3' positions. This is the first example of an enantioselective enzymatic reaction regulated by the design of metal complex in the protein active site.     

Solubilization of egg phosphatidylcholine liposomes by sodium taurocholate: partition behavior and morphology

Solubilization process of egg yolk phosphatidylcholine (EggPC) liposomes by sodium taurocholate (TC) was performed using membrane dialysis technique. Turbidity, apparent particle size, Cl⁻ permeability, membrane fluidity measured by electron spin resonance spectroscopy (ESR), and structural changes observed by freeze-fracture electron microscopy were investigated. The concentrations of TC were analyzed by high-performance liquid chromatography to determine the incorporation of TC in membrane phase and apparent partition coefficient. When TC concentration in water phase (D _w) was lower than 1.41 mM, or effective ratio of TC to EggPC (R _e) was less then 0.14, the partition coefficient was independent of the TC concentration and was 90 M⁻¹, where the membrane permeability of Cl⁻ was rather small. Upon increasing the concentrations in the range of 1.41 mM≤D _w<3.10 mM (0.14≤R _e<0.21), the apparent partition coefficient decreased and membrane permeability markedly increased. The coexistence of small vesicles and bilayer fragments were observed in this region. At 3.10 mM≤D _w<3.53 mM (0.21≤R _e<0.59), the steep decrease in turbidity was accompanied with the gradual increase in apparent partition coefficient, suggesting the formation of mixed micelles. The freeze-fracture electron micrographs confirmed the transformation of saturated bilayers to wormlike mixed micelles. When a further increase in TC (D _w≥3.53 or R _e≥0.59) was applied, the turbidity and apparent particle sizes became small. Transformation of the wormlike structure to ellipsoid mixed micelles was observed. The ESR spectra showed that TC/EggPC mixed micelles still retain a somewhat orderly structure.     

Diffuse reflectance infrared Fourier transform spectral study of the interaction of 3-aminopropyltriethoxysilane on silica gel. Behavior of amino groups on the surface

 Three silica gel sample systems, modified with 3-amino-propyltriethoxysilane (APTS), were prepared by sequentially sampling the reaction mixture at various time intervals, and the diffuse reflectance infrared Fourier transform (DRIFT) spectra of these samples were measured in the regions 2700–3500 and 1300–2000 cm^-1. The IR bands observed at 1597 and 1629–1633 cm^-1 were assigned to the deformation modes of NH₂ and NH⁺ ₃ groups, respectively. The intensities of these two bands are dependent on both the APTS concentration used in the preparation and the reaction time. The results are summarized as fol-lows. For the sample systems in which smaller APTS concentration were used, most of the NH₂ groups of the aminopropyl segments are converted into the NH⁺ ₃ groups on the surface, showing that the SiO^-…H⁺NH₂-type structure is predominantly stabilized on the surface of the silica gel. As the APTS concentration in the reaction mixture increases, the population of NH₂ groups in the silane layer coated onto the surface increases. Interpretation of the CH stretch region further suggests that cyclic structures may be formed on the surface as a consequence of the formation of NH⁺ ₃ groups. Received: 18 November 1996 Accepted: 14 February 1997     

Physical properties of phosphatidylcholine vesicles containing small amount of sodium cholate and consideration on the initial stage of vesicle solubilization

The effects of sub-solubilizing concentrations of sodium cholate (Na-chol) on several physicochemical properties of phosphatidylcholine (PC) small unilamellar vesicles (SUV) were considered in connection with the initial stage of membrane solubilization. ESR spectra of 12-doxylstearic acid (12-DS) in phosphatidylcholine from egg yolk (EPC) or dimyristoylphosphatidylcholine (DMPC) SUV at low concentrations (insufficient to destroy the vesicles) of Na-chol were composed of two (a strongly immobilized and an additional weakly immobilized) immiscible components. The origin of the additional bands was phase separation which occurred in the hydrophobic parts of PC SUV in the presence of Na-chol. Differential scanning calorimetry measurements demonstrated that the mixed DMPC/Na-chol SUV possessed two (a sharp low-temperature and a broad high-temperature) endothermic peaks, which is consistent with the coexistence of two immiscible phases in the vesicular membranes. zeta Potentials of the EPC/Na-chol SUV revealed that high anionic densities appeared on the surfaces of the SUV at a Na-chol concentration slightly below the upper boundary of the vesicle region. Thus, the initial stage of the solubilization of PC SUV by Na-chol was caused by the aggregation of hydrophobic parts of PC membranes, followed by the occurrence of high anionic densities on the surfaces of the vesicles. The fact that removal of Na-chol from PC/Na-chol mixed systems preferentially resulted in the formation of small vesicles might originate from these anionic charges.     

Nonannihilation dynamics in an exothermic reaction-diffusion system with mono-stable excitability

We consider a 2-component excitable and diffusive system which describes a simple exothermic reaction process. In some parameter regime, there are two characteristics of travelling pulses of the system: (i) travelling pulses are planarly unstable; (ii) when two travelling pulses approach closely, they do not annihilate each other and repel like elastic objects. Under this situation, it is shown that ring patterns break down into complex patterns in 2-dimensions, which are totally different from those arising in the well-known excitable and diffusive system with the FitzHugh-Nagumo nonlinearity. (c) 1997 American Institute of Physics.     

On factor states of -algebras and their extensions

We obtain some results on the unique extension of (factor) states of -algebras which complement various existing results. Our results also lead to a class of -algebras whose states are -convex sums of factor states.

     

Most standard spheres have smooth one fixed point actions ofA5. II

This paper proves that all standard spheresS ⁿ of dimension n=6 orn 9 have smooth one fixed point actions of the alternating groupA ₅ on five letters.Partially supported by the Yukawa Foundation, and also by Grant-in-Aid for Scientific Research 01740048.     

Equivariant Surgery Theory: Deleting-Inserting Theorems of Fixed Point Manifolds on Spheres and Disks

The paper gives a tool to delete and insert fixed point manifolds for smooth actions of finite Oliver groups on spheres and disks. A similar result was already given in a joint article with E. Laitinen and K. Pawaowski for those of finite nonsolvable groups on spheres. It is useful in classifying smooth actions on spheres from the view point of fixed point data. The methods employed in the present paper are equivariant surgery and equivariant connected sum associated with elements in the Burnside ring. The idea of killing surgery obstructions is as follows: Let G be a finite group not of prime power order, C a contractible, finiteG CW complex, and an element in a K theoretic group arising as an obstruction class of geometric object f. It often holds that (1-[C])^m becomes trivial for large integers m where [C] is the element represented byC in the Burnside ring (G). One expects that the algebraic object (1 - [C])^m is realizable as the obstruction class of G connected sum of f's related to (1 - [C])^m. Since it is true for the case here, we can kill the obstruction by taking G connected sum off's     

Image contrast analysis of STM images of self-assembled dioctadecyl chalcogenides on graphite at the liquid-solid interface.

The structures of self-assembled monolayers of dioctadecyl selenide (CH3(CH2)17)2Se and dioctadecyl telluride (CH3(CH2)17)2Te, as well as dioctadecyl ether (CH3(CH2)17)2O and dioctadecyl sulfide (CH3(CH2)17)2S, on graphite at the liquid-solid interface were systematically investigated by scanning tunneling microscopy (STM). Both dioctadecyl selenide and telluride formed monolayer structures in which the tilt angle between the molecular axis of the alkyl chain and the lamellae axis was 90 degrees , while dioctadecyl ether assembled with a tilt angle of 60 degrees . Dioctadecyl sulfide was found to make two different self-assembled structures having tilt angles of 60 and 90 degrees . When selenide was embedded in ether compounds in mixed self-assembled monolayers, the alkyl chains of the selenide became blurred, implying that the alkyl chains in the monolayers were unstable. This is in contrast with the structure of co-adsorbed monolayers of the ether and sulfide compounds, where the images of all alkyl chains had high spatial resolution. For the co-adsorbed monolayers, the image contrast of chalcogen atoms was normalized compared with that of alkyl chains of the ether compound in the same image frame. The normalized image contrast was found to be independent of the measurement conditions involving tip shapes, having the following trend, Te>Se>S>C>O. The difference in the normalized image contrast among chalcogen atoms are discussed based on fundamental parameters like polarizability and atomic radii.