Fumihiko Fujii

The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1‐ATPase by the ε subunit

The chloroplast‐type F1 ATPase is the key enzyme of energy conversion in chloroplasts, and is regulated by the endogenous inhibitor ε, tightly bound ADP, the membrane potential and the redox state of the γ subunit. In order to understand the molecular mechanism of ε inhibition, we constructed an expression system for the α3β3γ subcomplex in thermophilic cyanobacteria allowing thorough investigation of ε inhibition. ε Inhibition was found to be ATP‐independent, and different to that observed for bacterial F1‐ATPase. The role of the additional region on the γ subunit of chloroplast‐type F1‐ATPase in ε inhibition was also determined. By single molecule rotation analysis, we succeeded in assigning the pausing angular position of γ in ε inhibition, which was found to be identical to that observed for ATP hydrolysis, product release and ADP inhibition, but distinctly different from the waiting position for ATP binding. These results suggest that the ε subunit of chloroplast‐type ATP synthase plays an important regulator for the rotary motor enzyme, thus preventing wasteful ATP hydrolysis.

Amphiphilic p-sulfonatocalix[4]arene-coated CdSe/ZnS quantum dots for the optical detection of the neurotransmitter acetylcholine

Water-soluble CdSe/ZnS (core-shell) semiconductor quantum dots surface-modified with tetrahexyl ether derivatives of p-sulfonatocalix[4]arene were synthesized for the optical detection of the neurotransmitter acetylcholine.

Calixarene-coated water-soluble CdSe–ZnS semiconductor quantum dots that are highly fluorescent and stable in aqueous solution

A simple method for the preparation of highly fluorescent and stable, water-soluble CdSe-ZnS quantum dots is reported using calix[4]arene carboxylic acids as surface coating agents; the coating of the surface with the calixarene and the conjugation of antibodies to the quantum dots are confirmed by fluorescence correlation spectroscopy.

Anoxia induces matrix shrinkage accompanied by an increase in light scattering in isolated brain mitochondria

It is important to monitor mitochondrial conditions, and light scattering (LS) measurements have been applied to the detection of morphological changes in mitochondria in vivo. Little is known about the morphological and LS responses of brain mitochondria to oxygen withdrawal, a critical factor in cell death. We have therefore investigated the morphological and LS responses of isolated brain mitochondria to anoxia. Anoxia induced an increase in LS, reflecting mitochondrial matrix shrinkage. This response was reversible, but was reduced by adding digitonin, which disrupted the outer membrane selectively. This suggested that integrity of the outer membrane was necessary for the matrix response. We further examined the effects of Mg2+ and ATP on the responses because both exist in cells and modulate the changes in matrix volume. Although Mg2+ and ATP reduced the rates of increase and decrease in LS, respectively, the magnitudes of the increases in LS caused by anoxia stayed at over 80% of the control level (no Mg2+) in the presence of Mg2+ and ATP. This suggested that the increase in LS occurred in cells containing Mg2+ and ATP during anoxia. In contrast, that caused by inhibitors of the electron transport chain was reduced to below 30% of the control level in the presence of Mg2+. The present in vitro study provides a basis for interpretation of LS signals from mitochondria in brain research during oxygen withdrawal.

Preparation and Characterization of Thiacalix[4]arene Coated Water-Soluble CdSe/ZnS Quantum Dots as a Fluorescent Probe for Cu2+ Ions

Highly fluorescent water-soluble CdSe/ZnS (core/shell) quantum dots (QDs) as a fluorescent Cu2+ ion probe were synthesized using thiacalix[4]arene carboxylic acid (TCC) as a surface coating agent. Hydrophobic trioctylphosphine oxide (TOPO) capped CdSe/ZnS QDs were overcoated with TCC in tetrahydrofuran at room temperature, and deprotonation of the carboxyl groups of TCC resulted in the formation of water-soluble QDs. The surface structure of the QDs was characterized by using transmission electron microscopy (TEM) and fluorescence correlation spectroscopy (FCS). TEM images showed that TCC-coated QDs were monodispersed with the particle size (core-shell moiety) of approximately 5 nm. Hydrodynamic diameter of the TCC-coated QDs was determined to be 8.9 nm by FCS, showing that the thickness of the surface organic layer of the QDs was approximately 2 nm. These results indicate that the surface layer of TCC-coated QDs forms a bilayer structure consisting of TOPO and TCC molecules. TCC-coated CdSe/ZnS QDs were highly fluorescent (quantum yield, 0.21) compared to the QDs surface-modified with mercaptoacetic acid and mercaptoundecanoic acid. Fluorescence of the TCC-coated QDs was effectively quenched by Cu2+ ions even in the presence of other transition metal ions such as Cd2+, Zn2+, Co2+, Fe2+, and Fe3+ ions in the same solution. The Stern-Volmer plot for the fluorescence quenching by Cu2+ ions showed a linear relationship up to 30 microM of Cu2+ ions. The ion selectivity of TCC-coated QDs was determined by measurements of fluorescence responses towards biologically important transition metal ions (50 microM) including Fe2+, Fe3+, Co2+>Zn2+, Cd2+. The fluorescence of TCC-coated QDs was almost insensitive to other biologically important ions such as Na+, K+, Mg2+, and Ca2+, suggesting that TCC-coated QDs can be used as a fluorescent Cu2+ ion probe for biological samples. A possible quenching mechanism by Cu2+ ions was also discussed on the basis of a Langmuir-type adsorption isotherm.

Detection of antigen protein by using fluorescence cross-correlation spectroscopy and quantum-dot-labeled antibodies.

Prion diseases including bovine spongiform encephalopathy (BSE) are fatal neurodegenerative diseases associated with ACHTUNGTRENNUNGunconventional proteinaceous agents. The development of a method for detecting prion proteins (PrP) for early and sensitive diagnosis is essential to prevent the spread of such diseases. Fluorescence cross-correlation spectroscopy (FCCS) was proposed by Eigen and Rigler in 1994 and first realized by Schwille et al. It has been used to detect the interaction between two molecular species in vitro and also ex vivo. More recently, we applied FCCS to determine quantitatively the concentration of PrP. PrP was labeled with two fluorescent monoclonal antibodies (mAbs) against different epitopes, and the doubly labeled PrP was detected by FCCS with two laser lines (TL-FCCS). The sensitivity was comparable to that of enzyme-linked immunosorbent assay (ELISA), approved by the European Commission for BSE diagnosis. A perfect and stable overlap of two laser lines is often difficult; this reduces the amplitude and sensitivity of fluorescence cross-correlation functions (FCCFs). Thus we have developed a novel detection method using FCCS with semiconductor quantum dots (QDs). Because of QDs have broad absorption spectra, FCCS could be performed with single laser line (SL-FCCS). In this study, two kinds of PrP-specific mAb 44B1 and mAb 72 were used to detect recombinant bovine PrP (rBoPrP). For TL-FCCS analysis (excitation wavelengths 473 and 635 nm), the mAbs 44B1 and 72 were labeled with Alexa Fluor-488 and Alexa Fluor-647, respectively (i.e. , 44B1-Alex488 and 72Alex647). In contrast, for SL-FCCS analysis (excitation wavelength 473 nm), the mAbs 44B1 and 72 were labeled with Alexa Fluor-488 and QD-655, respectively (i.e. , 44B1-Alex488 and 72-QD655). The sample containing 44B1-Alex488 and 72-Alex647 had fluorescence autocorrelation functions (FACFs) in both the green and red channels of TL-FCCS (Figure 1A), but not in the red channel of SL-FCCS (Figure 1B, arrow) because Alexa Fluor647 was not excited by the 473 nm laser line (Figure 1B, top panel). In contrast, the sample containing 44B1-Alex488 and 72-QD655 had FCCFs in both channels of both TLand SLFCCS (Figures 1C and D, arrow), because QD-655 was also excited by 473 nm laser line (Figure 1D, top panel). The obtained diffusion times of Cy5 (fluorescent dye), 72-Alex647, and 72QD655 were 0.11 0.01, 0.90 0.04, and 2.36 0.36 ms, respectively. Since the size of the fluorescent dye and antibody are, respectively, about 1 and 10 nm, the hydrodynamic diameter of 72-QD655 was estimated to be about 25 nm. Using these pairs of labeled mAbs, we detected rBoPrP by FCCS in solution (Figure 2). rBoPrP can be detected in the presence of 44B1-Alex488 and 72-Alex647 by TL-FCCS (Figure 2A and B, arrow), also in the presence of 44B1-Alex488 and 72QD655 by SL-FCCS (Figures 2C and 2D, arrow). The y-intercept of FCCFs in SL-FCCS was higher than that in TL-FCCS. This was probably caused by the excitation of the sample by the single laser line in SL-FCCS. Since there was no cross correlation at 0 nm rBoPrP (Figures 2A and C), we consider that the crosstalk was free or very week in our experimental conditions. Figure 3 shows FACFs in the green channel and FCCFs at various concentrations of rBoPrP with the two pairs of mAbs, 72-Alex488/44B1-Alexa647 (A and B) and 44B1-Alex488/72QD655 (C and D). FACFs in the green channel shifted to the right only with the 44B1-Alex488 and 72-QD655 pair (Figure 3A and C) because QD655 was a heavy tag, but Alex647 was not. On the other hand, the amplitude of the FCCFs rose with increasing rBoPrP concentration in both samples (FigACHTUNGTRENNUNGures 3B and D). It is noteworthy that the amplitude of FCCFs in SL-FCCS was remarkably higher than that in TL-FCCS at ACHTUNGTRENNUNGvarious concentrations of rBoPrP. The concentration of the immune complex was at a maximum at 0.232 nm rBoPrP. The counts per molecule (brightness per molecule) of 72-QD655 at 0 and 0.232 nm rBoPrP were 15.4 1.7 and 17.4 2.0 kHz, respectively. Since there is no significant difference between them, FRET was negligible under our experimental conditions. To allow a more accurate comparison between the samples, the amplitude of FCCFs was normalized against that of FACFs in the green channel, that is, Cc(0)/Cg(0) (Figure 4, see Experimental Section). The ratios, Cc(0)/Cg(0), of the samples containing 44B1-Alex488 and 72-QD655 were higher than those of the samples containing 44B1-Alex488 and 72-Alex647, for example by a factor of 5 at 0.927 nm rBoPrP. When the cutoff line was defined as the mean plus 3 standard deviations of the sample without rBoPrP, the detection limit of the pairs of 44B1-Alex488/72-QD655 and 44B1-Alex488/72-Alex647 were 0.029 and 0.37 nm, respectively (Table 1). The method using SL-FCCS with QDs was about ten times more sensitive than that using TL-FCCS with Alexa dyes. In addition, the detection limit of ELISA (Bio-Rad, USA) approved by the European Commission for BSE diagnosis was 0.15 nm in PBS (pH 7.3; Table 1), which was about five times higher than the method using SLFCCS with QD. Since FCS and FCCS analyses require only microliter samples without any separation steps, the methods are suitable for high-throughput screening with high sensitivity. Stoevesandt et al. have already demonstrated the testing of inhibitors of protein complexes in crude cell lysates using both FCS and FCCS. Their methods consist of two independent sample preparation and measurement procedures such as fluorophore labeling and heavy tagging for FCS, and labeling with two [a] Dr. F. Fujii, Prof. Dr. M. Kinjo Laboratory of Molecular Cell Dynamics Faculty of Advanced Life Science Graduate School of Life Science, Hokkaido University Sapporo 001-0021 (Japan) Fax: (+81)11-706-9006 E-mail : Kinjo@imd.es.hokudai.ac.jp

Three Dimensional Orientation Measurements of Single Fluorescent Molecules by Newly Developed Polarized Fluorescence Microscopy

Single molecule fluorescence techniques are increasingly important to observe the dynamic properties of single molecules. One such important dynamic property is the single molecules orientation. In order to observe three dimensional motions of proteins in solution, it is necessary to measure three dimensional orientations of proteins. We developed new microscopy for determining three dimensional orientations based on the principal of polarization analysis proposed by Fourkas. This method requires only that one collect fluorescence counts from a single molecule at three different polarizations followed by a simple mathematical calculation to yield the three dimensional orientations. In this method, the relatively small numbers of photons are sufficient for a reliable orientation measurement and this should decrease the time scale needed to determine the orientation of any given fluorophore. Here, we demonstrate axial rotation of actin filaments sliding over myosin molecules fixed on a glass surface by polarization measurement of individual rhodamine phalloidin fluorophores sparsely bound to filaments. This new microscopy will be available for investigating the wide range of dynamic processes through single molecule orientation dynamics in various biophysical studies.

Detection of recombinant bovine prion protein by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a detection technique for molecular parameters, such as concentration, diffusion constant and interaction in solution with single molecule level. FCS consists of a confocal optics with a well focused laser beam and detects fluorescent fluctuation that is caused by the diffusion of fluorescently labeled molecules through a tiny open volume element (< one femtoliter). Measured fluctuation can be used to calculate the lateral diffusion coefficient and thus the size of the molecule. We propose FCS as a newly method for the detection of prion diseases.

The Reaction of Copper in Cytochrome Oxidase with Cytochrome C in Rat Brain In Situ

The role of copper of cytochrome oxidase in the electron transfer sequence proposed in vitro was investigated by spectroscopically monitoring the steady-state redox changes of cytochrome c, copper and heme a + a3 of cytochrome oxidase in the perfused rat head. The percent oxidation of copper and cytochrome c in the aerobic-anaerobic transition did not show a straight-line relationship. As compared with copper, cytochrome c was most reduced, followed by heme a + a3. These results suggested that copper first accepted an electron from heme a rather than cytochrome c in the mitochondrial membrane, unlike the electron transfer sequence proposed for purified enzymes. The orientation between cytochrome c and cytochrome oxidase would appear to be controlled primarily by the membrane structure.