Biomedical subjects

Yoshiyuki Amemiya

Publications and source records attributed to Yoshiyuki Amemiya.

10 recordsLinked to original sources

PubMed2006

A 3 x 6 arrayed CCD X-ray detector for continuous rotation method in macromolecular crystallography.

A 3 x 6 arrayed charge-coupled device (CCD) X-ray detector has been developed for the continuous-rotation method in macromolecular crystallography at the Photon Factory. The detector has an area of 235.9 mm x 235.9 mm and a readout time of 1.9 s. The detector is made of a 3 x 6 array of identical modules, each module consisting of a fiber-optic taper (FOT), a CCD sensor and a readout circuit. The outputs from 18 CCDs are read out in parallel and are then digitized by 16-bit analog-to-digital converters. The advantage of this detector over conventional FOT-coupled CCD detectors is the unique CCD readout scheme (frame transfer) which enables successive X-ray exposures to be recorded without interruption of the sample crystal rotation. A full data set of a lysozyme crystal was continuously collected within 360 s (180 degrees rotation, 3 s/1.5 degrees frame). The duty-cycle ratio of the X-ray exposure to the data collection time was almost 100%. The combination of this detector and synchrotron radiation is well suited to rapid and continuous data collection in macromolecular crystallography.

Crystallography, X-Ray↗

PubMed2006

Structural analysis of human hair single fibres by scanning microbeam SAXS.

The origin of the curliness of human hair was revealed by scanning microbeam small angle X-ray scattering (SAXS), based on the nanostructure of keratin fibre arrangement. Scanning microbeam SAXS patterns of single hair fibres have been measured across the fibres and the differences in the patterns between the inner and the outer sides of the curvature were successfully detected. The analysis of the equatorial and azimuthal scattering intensity profiles showed that the arrangement of the intermediate filaments was different between the inner and the outer sides of the curvature. From the analogy with Merino and Romny wool, it is suggested that different types of cortices exist in human hair. It is concluded that, regardless of the ethnic origins (African, Caucasian, and Asian), the macroscopic curl shape of the hair fibre originate from the inhomogeneity of the internal nanostructure, arising from inhomogeneous distribution of two types of cortices.

Black or African American↗

PubMed2005

Helical and expanded conformation of equine beta-lactoglobulin in the cold-denatured state.

The thermal unfolding transition of equine beta-lactoglobulin (ELG) was investigated by circular dichroism (CD) over a temperature range of -15 degrees C to 85 degrees C. In the presence of 2 M urea, a cooperative unfolding transition was observed both with increasing and decreasing temperature. The CD spectrum indicated that the heat and cold-denatured states of ELG have substantial secondary structures but lack persistent tertiary packing of the side-chains. In order to clarify the relation between the heat or cold-denatured state and the acid-denatured (A) state characterized previously, we have attempted to observe the temperature dependence of the CD spectrum at pH 1.5. The CD spectrum in the heat-denatured state is similar to that in the A state. The CD spectrum in the A state does not change cooperatively with increasing temperature. These results indicate that the heat-denatured state and the A state are the same structural state. On the other hand, the CD intensity at acid pH cooperatively increased with decreasing temperature. The CD spectrum at low temperature and acid pH is consistent with that in the cold-denatured state. Therefore, the cold-denatured state is distinguished from the heat-denatured state or the A state, and ELG assumes a larger amount of non-native alpha-helices in the cold-denatured state. Small angle X-ray scattering and analytical ultracentrifugation have indicated that ELG assumes an expanded chain-like conformation in the cold-denatured state in contrast to the compact globular conformation in the A state. The relation between the molecular size and the helical content in the partially folded states is discussed.

Animals↗

PubMed2005

Influence of nucleotide effectors on the kinetics of the quaternary structure transition of allosteric aspartate transcarbamylase.

We report the effects of allosteric effectors, ATP, CTP and UTP on the kinetics of the quaternary structure change of Escherichia coli ATCase during the enzyme reaction with physiological substrates. Time-resolved, small-angle, X-ray scattering of solutions allows direct observation of structural transitions over the entire time-course of the enzyme reaction initiated by fast mixing of the enzyme and substrates. In the absence of effectors, all scattering patterns recorded during the reaction are consistent with a two-state, concerted transition model, involving no detectable intermediate conformation that differs from the less active, unliganded T-state and the more active, substrate-bound R-state. The latter predominates during the steady-state phase of enzyme catalysis, while the initial T-state is recovered after substrate consumption. The concerted character of the structural transition is preserved in the presence of all effectors. CTP slightly shifts the dynamical equilibrium during a shortened steady state toward T while the additional presence of UTP makes the steady state vanishingly short. The return transition to the T conformation is slowed significantly in the presence of inhibitors, the effect being most severe in the presence of UTP. While ATP increases the apparent T to R rate, it also increases the duration of the steady-state phase, an apparently paradoxical observation. This observation can be accounted for by the greater increase in the association rate constant of aspartate, promoted by ATP, while the nucleotide produces a lesser degree of increase in the dissociation rate constant. Under our experimental conditions, using high concentrations of both enzyme and substrate, it appears that this very mechanism of activation turns the activator into an efficient inhibitor. The scattering patterns recorded in the presence of ATP support the view that ATP alters the quaternary structure of the substrate-bound enzyme, an effect reminiscent of the reported modification of PALA-bound R-state by Mg-ATP.

Allosteric Regulation↗

PubMed2005

Observation of the transient rotator phase of n-hexadecane in emulsified droplets with time-resolved two-dimensional small- and wide-angle X-ray scattering.

Crystallization of n-hexadecane in emulsion droplets was studied using time-resolved two-dimensional small- and wide-angle x-ray scattering with differential scanning calorimetry (2D-SAXS-WAXS-in situ DSC) which provides information about both nano- and subnanoscale structural change. n-hexadecane in droplets reproducibly crystallized into the stable triclinic phase via a transient-rotator phase. This is in contrast with previous results that the rotator phase of n-hexadecane was observed only occasionally for bulk samples. Thus we confirmed the existence of rotator phase in n-hexadecane, which is important for the study of crystallization of soft materials. We suggest that the rotator phase at the interface of oil and water plays a precursor role for bulk crystallization. This study demonstrates that 2D-SAXS-WAXS-in situ DSC is a powerful tool for the study of a transient phase.

Journal Article↗

PubMed2003

Equilibrium and kinetics of the allosteric transition of GroEL studied by solution X-ray scattering and fluorescence spectroscopy.

We have studied the ATP-induced allosteric structural transition of GroEL using small angle X-ray scattering and fluorescence spectroscopy in combination with a stopped-flow technique. With X-ray scattering one can clearly distinguish the three allosteric states of GroEL, and the kinetics of the transition of GroEL induced by 85 microM ATP have been observed directly by stopped-flow X-ray scattering for the first time. The rate constant has been found to be 3-5s(-1) at 5 degrees C, indicating that this process corresponds to the second phase of the ATP-induced kinetics of tryptophan-inserted GroEL measured by stopped-flow fluorescence. Based on the ATP concentration dependence of the fluorescence kinetics, we conclude that the first phase represents bimolecular non-cooperative binding of ATP to GroEL with a bimolecular rate constant of 5.8 x 10(5)M(-1)s(-1) at 25 degrees C. Considering the electrostatic repulsion between negatively charged GroEL (-18 of the net charge per monomer at pH 7.5) and ATP, the rate constant is consistent with a diffusion-controlled bimolecular process. The ATP-induced fluorescence kinetics (the first and second phases) at various ATP concentrations (< 400 microM) occur before ATP hydrolysis by GroEL takes place and are well explained by a kinetic allosteric model, which is a combination of the conventional transition state theory and the Monod-Wyman-Changeux model, and we have successfully evaluated the equilibrium and kinetic parameters of the allosteric transition, including the binding constant of ATP in the transition state of GroEL.

Adenosine Triphosphate↗

PubMed2003

Kinetic studies of unfolding process of aspergillopepsin II by pH-jump methods.

pH-induced unfolding process of the acid proteinase aspergillopepsin II was studied kinetically by stopped-flow methods using circular dichroism and small angle X-ray scattering. Native aspergillopepsin II consists of two polypeptide chains, a light chain and a heavy chain, which are bound noncovalently to each other at acidic pH. Above neutral pH the two chains are known to dissociate and swell due to electrostatic repulsion inside the molecule. The present studies suggested that the polypeptide chains dissociate first and that the dissociated heavy chain expands subsequently. This two-phased pathway is consistent with the accumulation of an intermediate species in the pH-induced unfolding of the enzyme.

Aspartic Acid Endopeptidases↗

PubMed2003

Denaturation and reassembly of chaperonin GroEL studied by solution X-ray scattering.

We measured the denaturation and reassembly of Escherichia coli chaperonin GroEL using small-angle solution X-ray scattering, which is a powerful technique for studying the overall structure and assembly of a protein in solution. The results of the urea-induced unfolding transition show that GroEL partially dissociates in the presence of more than 2 M urea, cooperatively unfolds at around 3 M urea, and is in a monomeric random coil-like unfolded structure at more than 3.2 M urea. Attempted refolding of the unfolded GroEL monomer by a simple dilution procedure is not successful, leading to formation of aggregates. However, the presence of ammonium sulfate and MgADP allows the fully unfolded GroEL to refold into a structure with the same hydrodynamic dimension, within experimental error, as that of the native GroEL. Moreover, the X-ray scattering profiles of the GroEL thus refolded and the native GroEL are coincident with each other, showing that the refolded GroEL has the same structure and the molecular mass as the native GroEL. These results demonstrate that the fully unfolded GroEL monomer can refold and reassemble into the native tetradecameric structure in the presence of ammonium sulfate and MgADP without ATP hydrolysis and preexisting chaperones. Therefore, GroEL can, in principle, fold and assemble into the native structure according to the intrinsic characteristic of its polypeptide chain, although preexisting GroEL would be important when the GroEL folding takes place under in vivo conditions, in order to avoid misfolding and aggregation.

Chaperonin 60↗

PubMed2002

Fast compaction of alpha-lactalbumin during folding studied by stopped-flow X-ray scattering.

To monitor the fast compaction process during protein folding, we have used a stopped-flow small-angle X-ray scattering technique combined with a two-dimensional charge-coupled device-based X-ray detector that makes it possible to improve the signal-to-noise ratio of data dramatically, and measured the kinetic refolding reaction of alpha-lactalbumin. The results clearly show that the radius of gyration and the overall shape of the kinetic folding intermediate of alpha-lactalbumin are the same as those of the molten globule state observed at equilibrium. Thus, the identity between the kinetic folding intermediate and the equilibrium molten globule state is firmly established. The present results also suggest that the folding intermediate is more hydrated than the native state and that the hydrated water molecules are dehydrated when specific side-chain packing is formed during the change from the molten globule to the native state.

Animals↗

PubMed2002

X-ray four-quadrant diamond phase-retarder system to compensate for off-axis and chromatic aberrations.

An X-ray transmission-type double phase-retarder system that can compensate for off-axis aberration (phase-shift inhomogeneity due to angular divergence of incident X-rays) has been developed and its advantage over a conventional single transmission-type X-ray phase retarder was demonstrated. However, it was noticed that the transmission-type X-ray phase retarder suffers from not only off-axis aberration but also chromatic aberration (phase-shift inhomogeneity due to energy spread of incident X-rays). In this paper, a transmission-type X-ray four-quadrant phase-retarder system is proposed that can compensate for both off-axis and chromatic aberrations. The X-ray four-quadrant phase-retarder system is composed of four transmission-type X-ray phase retarders. The scattering planes of four phase-retarder diamond crystals are set to be inclined by 45 degree, 135 degree (= 45 degree + 90 degree), 225 degree (= 45 degree + 180 degree) and 315 degree (= 45 degree + 270 degree), respectively, with respect to the plane of incident polarization. Under the conditions of 7709 eV photon energy, 1.5 eV energy spread and 45" angular divergence (FWHM) of incident X-rays, the X-ray four-quadrant phase-retarder system created 0.98 degree of vertical-linear polarization from horizontal-linear polarization. This value was favorably compared to 0.89 and 0.96, which were obtained by the single and double phase-retarder systems, respectively. The principle of the X-ray four-quadrant phase-retarder system will be described together with its advantage over the single and double phase-retarder systems.

Journal Article↗