Yohko F. Yano

Kinetics of protein unfolding at interfaces

The conformation of protein molecules is determined by a balance of various forces, including van der Waals attraction, electrostatic interaction, hydrogen bonding, and conformational entropy. When protein molecules encounter an interface, they are often adsorbed on the interface. The conformation of an adsorbed protein molecule strongly depends on the interaction between the protein and the interface. Recent time-resolved investigations have revealed that protein conformation changes during the adsorption process due to the protein-protein interaction increasing with increasing interface coverage. External conditions also affect the protein conformation. This review considers recent dynamic observations of protein adsorption at various interfaces and their implications for the kinetics of protein unfolding at interfaces.

Driving force behind adsorption-induced protein unfolding: a time-resolved X-ray reflectivity study on lysozyme adsorbed at an air/water interface.

Time-resolved X-ray reflectivity measurements for lysozyme (LSZ) adsorbed at an air/water interface were performed to study the mechanism of adsorption-induced protein unfolding. The time dependence of the density profile at the air/water interface revealed that the molecular conformation changed significantly during adsorption. Taking into account previous work using Fourier transform infrared (FTIR) spectroscopy, we propose that the LSZ molecules initially adsorbed on the air/water interface have a flat unfolded structure, forming antiparallel beta-sheets as a result of hydrophobic interactions with the gas phase. In contrast, as adsorption continues, a second layer forms in which the molecules have a very loose structure having random coils as a result of hydrophilic interactions with the hydrophilic groups that protrude from the first layer.

Real-time investigation of protein unfolding at an air-water interface at the 1 s time scale

Protein unfolding at an air–water interface is followed in real time by a recently developed simultaneous multiple-angle–wavelength-dispersive X-ray reflectometer with a time resolution of 1 s.

A simultaneous multiple angle-wavelength dispersive X-ray reflectometer using a bent-twisted polychromator crystal

Using a convergent X-ray beam having continuously varying energy and glancing angle as a function of direction, the whole profile of a specular X-ray reflectivity curve is measured with no need for any mechanical motion during the measurement.

Hofmeister Anion Effects on Protein Adsorption at an Air–Water Interface

Hofmeister anion effects on adsorption kinetics of the positively charged lysozyme (pH < pI) at an air-water interface were studied by surface tension measurements and time-resolved X-ray reflectometry. In the salt-free solution, the protein adsorption rate increases with decreasing the net positive charge of lysozyme. When salt ions are dissolved in water, the protein adsorption rate drastically increases, and the rate is following an inverse Hoffmeister series (Br(-) > Cl(-) > F(-)). This is the result of the strongly polarized halide anion Br(-) being attracted to the adsorbed protein layer due to strong interaction with local electric field, while weakly polarized anion F(-) having no ability to penetrate the protein layer. In X-ray reflection studies, we observed that the lysozyme molecules initially adsorbed on the air-water interface have a flat unfolded structure as previously reported in the salt-free solution. In contrast, in the concentrated salt solutions, the lysozyme molecules begin to refold during adsorption. This protein refolding as a result of protein-protein rearrangements may be a precursor phenomenon of crystallization. The refolding is most significant for Cl(-), which is a good crystallization agent, whereas it is less observed for the strongly hydrated F(-). It is widely known in the bulk state that kosmotropic anions tend to precipitate proteins but at the same time stabilize proteins against denaturing. On the other hand, at the air-water interface where adsorbed proteins usually unfold, we observed chaotropic anions strongly bound to proteins that reduce electrostatic repulsion between protein molecules, and subsequently they induce protein refolding whereas the kosmotropic anions do not.

Use of Imaging Plates in Gas-Phase Electron Diffraction

In place of the photographic films used so far, imaging plates (IP) have been tried for use in gas-phase electron diffraction. With the exposure conditions suitable for photographic films, signal intensities of sufficient strength were obtained, and the results of a least-squares analysis for carbon disulfide showed that standard deviations of structural parameters were of similar magnitude as those obtained by photographic films. By virtue of the wide dynamic range of IP, data of similar quality were obtained even without rotating a sector.

A method for measuring the specular X-ray reflectivity with millisecond time resolution

A method for quick measurement of the specular X-ray reflectivity using a tapered undulator source is described. It uses a convergent X-ray beam with a one-to-one correspondence between X-ray energy and direction, which is produced by diffraction of a white X-ray beam at a curved silicon crystal. To increase the momentum transfer range, the sample is rotated 45? from the horizontal around the incident beam direction, so that both the X-ray energy and the incident angle change continuously with direction. The specularly reflected beam is observed with a two-dimensional detector. The X-ray reflectivity curve in a wide momentum transfer range can be observed in a single detector exposure with a time resolution in the millisecond range. Test measurements were done for a commercial silicon wafer and a gold thin film on silicon.

Quick X-Ray Reflectometry in the Simultaneous Multiple Angle-Wavelength Dispersive Mode

The whole profile of the specular X-ray reflectivity curve was simultaneously and quickly measured with no need to rotate the specimen, the detector or the monochromator crystal. A white synchrotron beam from a bending magnet source is incident on a bent-twisted silicon (111) crystal polychromator that produces a convergent X-ray beam with a continuously varying wavelength (energy) and glancing angle to the specimen surface. This convergent X-ray beam was specularly reflected in the vertical direction by the specimen placed at the focus. The normalized spatial distribution across the beam direction of the reflected beam represents a specular X-ray reflectivity curve because each position along the line recorded on the two dimensional detector surface corresponds to a different momentum transfer. Reflectivity curves from a (001) silicon wafer, a nickel thin film on a silicon substrate, and a water surface were measured with data collection times of 0.001-100 s, 0.01-100 s, and 1.0-1000 s, respectively. The simultaneously covered momentum transfer range was 0.03-0.52 A−1 for solid specimens and 0-0.41 A−1 for liquid specimen.

A quick convergent-beam laboratory X-ray reflectometer using a simultaneous multiple-angle dispersive geometry

An X-ray reflectometer, using a laboratory X-ray source, that can measure the specular X-ray reflectivity curve with a time resolution of 10 s or less was developed. Low reflectivities can be measured accurately because the background can be subtracted from the signal.

Structure of Liquid Chloroform as Investigated by Energy-Dispersive X-Ray Diffraction

The structure of liquid chloroform has been investigated by energy-dispersive X-ray diffraction. For the stability of the sample thickness, even for a volatile liquid, a glass capillary tube has been used as a sample container. The intensity function has been accounted for by a bcc (body-centered cubic)-like local lattice structure model with a lattice constant of 6.24 A. However, it has been necessary to replace a fraction (34%) of the nearest neighbors by super-near configurations in which molecules are rather tightly correlated. The correlation is, however, rapidly lost as the distance between molecules increases, and the long range structure is characterized by a large Prins parameter. The structure of liquid chloroform is much less ordered than that of carbon tetrachloride.The structure of liquid chloroform has been investigated by energy-dispersive X-ray diffraction. For the stability of the sample thickness, even for a volatile liquid, a glass capillary tube has been used as a sample container. The intensity function has been accounted for by a bcc (body-centered cubic)-like local lattice structure model with a lattice constant of 6.24 A. However, it has been necessary to replace a fraction (34%) of the nearest neighbors by super-near configurations in which molecules are rather tightly correlated. The correlation is, however, rapidly lost as the distance between molecules increases, and the long range structure is characterized by a large Prins parameter. The structure of liquid chloroform is much less ordered than that of carbon tetrachloride.