Structures of Nanoconfined Liquids Determined by Synchrotron X-ray Diffraction.

We have successfully performed X-ray diffraction measurements of the liquids octamethylcyclotetrasiloxane (OMCTS, a quasi-spherical-shaped molecule) and n-hexadecane (a normal alkane) confined between mica surfaces at surface separation distances (D's) from 500 nm to the hard-wall thickness (1.9 nm for OMCTS and 1.0 nm for hexadecane). At all of the studied D's, we observed diffraction peaks corresponding to their mean intermolecular spacing at q = 8.6 nm-1 (d = 0.73 nm) for OMCTS and q = 13.6 nm-1 (d = 0.45 nm) for n-hexadecane. The peak intensity increased at D < ca. 50 nm for OMCTS even with the decreasing distance and exhibited a local maximum at D = 17-13 nm, indicating the sharp increase in the molecular order in this distance range. The peak intensities normalized by the D and Inormalized values of OMCTS and n-hexadecane were nearly constant at D's greater than 100 nm, though they appeared to increase slightly. The increase then became more significant with decreasing D below 100 nm, and finally the Inormalized values became 120 (for OMCTS) and 160 (for n-hexadecane) at the hard wall. These results clearly demonstrated the significant increase in the structural order of OMCTS and n-hexadecane under nanoconfinement, especially below 100 nm. The fwhm values of the peaks of OMCTS and n-hexadecane showed no significant change until small distances when the confinement effect was significant. These results indicated that the increase in the structural order should be mainly ascribed to the ordering of the molecules in the parallel plane in the enhanced layered structure formed under the confinement. The viscous parameters (b2) of OMCTS and n-hexadecane obtained from the resonance shear measurement showed no increase at D's down to ca. 7 nm. This indicated that a certain ordering of the confined molecules was required for the observable increase in the viscosity.

Effects of surface and shear forces on nano-confined smectic-A liquid crystals studied by X-ray diffraction.

The orientational behavior of a smectic-A liquid crystal (4-cyano-4'-octylbiphenyl, 8CB) confined between mica surfaces as well as between silica surfaces with a nanometer scale thickness was investigated by synchrotron X-ray diffraction measurement. The crystallographic axes of two confining mica sheets were adjusted parallel to each other to induce the preferential orientation of 8CB molecules along their crystallographic axis. The silica surfaces, which were hydrophilic and amorphous and had nanometer level smoothness, were prepared on mica surfaces using a sputtering technique. The X-ray diffraction measurement revealed that the 8CB molecules, confined between mica surfaces (DHW = 1.7 nm) and between silica surfaces (DHW = ca. 2 nm), took a planar orientation (oriented its long axis parallel to the surface) and formed a lamellar structure. However, the in-plane orientation of the confined 8CB changed depending on the confining surfaces. The lamellar axis of the 8CB confined between mica surfaces uniaxially oriented most probably due to the preferential alignment of its long axis along the principal crystallographic a-axis of the mica. On the other hand, 8CB between the silica surfaces formed lamellar domains in which the lamellar axis of 8CB omnidirectionally oriented in-plane. The effect of the shear on the orientation of the nano-confined 8CB was also investigated. The lamellar axis, corresponding to the long axis of the 8CB molecules confined between the mica surfaces, rotated only ca. 3 degrees within the plane parallel to the surface by perpendicularly applying shear to the axis. The lamellar axis of the 8CB molecules between the silica surfaces showed no noticeable change by applying the shear. These results indicated that the effect of shear to align the 8CB molecules was significantly suppressed due to the confinement effect which significantly reduces the mobility of molecules as well as the alignment effect along the crystallographic axis in the case of mica. We also observed a change in the orientation of nano-confined 8CB after shear treatment at large D (= 3.3 µm).

Differential Scanning Calorimetry Study on the Adsorption of Myoglobin at Mesoporous Silicas: Effects of Solution pH and Pore Size.

In the present study, pore adsorption behavior of globular myoglobin (Mb) at mesoporous silicas was examined utilizing the low-temperature differential scanning calorimetry (DSC) method. The DSC method relies on a decrease in heat of fusion for the pore water upon adsorption of Mb. The amount and structure of Mb adsorbed into the mesoporous silica were examined by DSC and optical absorption spectroscopy. The results indicated that the pore adsorption behavior of Mb strongly depended on the solution pH and pore size of mesoporous silica. For the adsorption of Mb (diameter = 3.5 nm) into mesoporous silica with narrow pores (pore diameter = 3.3 nm) at a pH ranging from 7.0 to 3.7, the penetration of both folded and denatured Mb molecules was confirmed. The folded Mb could penetrate into large mesoporous silica pores (pore diameter = 5.3 and 7.9 nm), whereas the penetration of the denatured Mb molecules was completely inhibited. The distribution of folded Mb at mesoporous silica depended on the pore size; almost all folded Mb molecules located inside mesoporous silica pores of diameters 3.3 and 5.3 nm, whereas the Mb molecules distributed at bot internal and external pore surfaces of mesoporous silica with 7.9 nm in pore diameter. These pore adsorption behaviors suggest that aggregation or stacking of the Mb molecules at the pore entrance regions of the large pores affected the pore adsorption behavior.

Continuous Mesoporous Aluminum Oxide Film with Perpendicularly Oriented Mesopore Channels.

Mesoporous aluminum oxide (MAO) films with perpendicularly oriented cylindrical mesopores (pore diameter: ca. 10 nm) were successfully deposited on a glass substrate by a surfactant-templating approach using aluminum nitrate as an aluminum source. The perpendicular orientation of mesopores was confirmed by grazing-incidence small-angle X-ray scattering and neutron reflection experiments. The thickness of the MAO film was around 100 nm, with a surface roughness of less than 6 nm. Since the inner surface of MAO pores was positively charged, negatively charged glucose oxidase molecules could be densely loaded into the cylindrical mesopores without significant loss of enzymatic activity. The present MAO film is potentially useful as an inorganic host material for an enzyme toward the development of a biocatalytic system.

Characterization of Myoglobin Adsorption into Mesoporous Silica Pores by Differential Scanning Calorimetry.

Adsorption of protein molecules into the pores of a porous material is an important process for chromatographic separation of proteins and synthesis of nanoscale biocatalyst systems; however, there are barriers to developing a method for analyzing the process quantitatively. The purpose of this study is to examine the applicability of differential scanning calorimetry (DSC) for quantitative analysis of protein adsorption into silica mesopores. For this purpose myoglobin, a globular protein (diameter: 35.2 Å) was selected, and its adsorption onto mesoporous silica powders with uniform pore diameters (pore diameters: 39 and 64 Å) was measured by adsorption assay and DSC experiments. Our results confirmed that the adsorption of myoglobin into the silica mesopores induced significant changes in the positions and areas of freezing/melting peaks of the pore water. The decrease in heat of fusion of the pore water after myoglobin adsorption could be utilized to quantify the amount of myoglobin inside the silica mesopores. The advantages of DSC include its applicability to small wet mesoporous silica samples.

Effect of Cavity Size of Mesoporous Silica on Short DNA Duplex Stability.

We studied the stabilities of short (4- and 3-bp) DNA duplexes within silica mesopores modified with a positively charged trimethyl aminopropyl (TMAP) monolayer (BJH pore diameter 1.6-7.4 nm). The DNA fragments with fluorescent dye were introduced into the pores, and their fluorescence resonance energy transfer (FRET) response was measured to estimate the structuring energies of the short DNA duplexes under cryogenic conditions (temperature 233-323 K). The results confirmed the enthalpic stability gain of the duplex within size-matched pores (1.6 and 2.3 nm). The hybridization equilibrium constants found for the size-matched pores were 2 orders of magnitude larger than those for large pores (≥3.5 nm), and this size-matching effect for the enhanced duplex stability was explained by a tight electrostatic interaction between the duplex and the surface TMAP groups. These results indicate the requirement of the precise regulation of mesopore size to ensure the stabilization of hydrogen-bonded supramolecular assemblies.

Probing structure-function relationships in early events in photosynthesis using a chimeric photocomplex.

The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium Thermochromatium tepidum requires Ca2+ for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the Tch. tepidum LH1 complex in an engineered Rhodobacter sphaeroides mutant strain. This system contained a chimeric pufBALM gene cluster (pufBA from Tch. tepidum and pufLM from Rba. sphaeroides) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed Tch. tepidum wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in Rba. sphaeroides) was incorporated into the Tch. tepidum LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αß-subunit. The hybrid LH1-RC complexes expressed in Rba. sphaeroides were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, ß-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the Tch. tepidum LH1 complex; in contrast, α-N50 does not participate in Ca2+ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated Tch. tepidum LH1-RC complex and have unambiguously identified the location of Ca2+ within this key antenna complex.

Thermodynamics of Complexation between Thiourea-based Receptor and Acetate in Water/Acetonitrile Mixture.

A thiourea-based receptor has been extensively studied for selective anion recognition for reasons of its strong hydrogen bond donor ability. In the present study, the thermodynamics of complexation between a thiourea-based receptor and acetate was examined in a water/acetonitrile mixture. The receptor used in this study was N,N'-bis(p-nitrophenyl)thiourea (BNPTU). UV/vis spectroscopic titration and isothermal titration calorimetry (ITC) experiments clearly revealed endothermic and entropy-driven complexation of BNPTU with acetate in water/acetonitrile mixtures. Since the endothermic peaks found in water/acetonitrile mixtures were about three times greater than those in acetonitrile, it appears that preferential hydration of both receptor and acetate was responsible for the endothermic and entropy-driven complexation reaction. The thermodynamic properties found in this study have the potential to contribute to the design of a thiourea-based anion receptor.

Structural Stability of Light-harvesting Protein LH2 Adsorbed on Mesoporous Silica Supports.

In the present study, we examined the reversible thermal deformation of the membrane protein light-harvesting complex LH2 adsorbed on mesoporous silica (MPS) supports. The LH2 complex from Thermochromatium tepidum cells was conjugated to MPS supports with a series of pore diameter (2.4 to 10.6 nm), and absorption spectra of the resulting LH2/MPS conjugates were observed over a temperature range of 273 - 313 K in order to examine the structure of the LH2 adsorbed on the MPS support. The experimental results confirmed that a slight ellipsoidal deformation of LH2 was induced by adsorption on the MPS supports. On the other hand, the structural stability of LH2 was not perturbed by the adsorption. Since the pore diameter of MPS support did not influence the structural stability of LH2, it could be considered that the spatial confinement of LH2 in size-matches pore did not improve the structural stability of LH2.