Hiroshima Synchrotron Radiation Center Hiroshima University

2-13. Vacuum-ultraviolet circular dichroism spectroscopy beamline (BL15)

Circular dichroism (CD) spectroscopy is widely used for the structural analysis of optically active materials such as proteins, sugars, and DNA. However, CD measurement in the vacuum-ultraviolet (VUV) region below 190 nm is generally difficult using conventional CD spectrophotometers because of some technical difficulties in the light source, optical device, and sample cell. The extension of CD measurements into the VUV region could provide more detailed and new information on the structure of biomolecules based on the higher energy transitions of chromophores such as hydroxyl and acetal groups. Since the 1980s, several facilities have devoted considerable effort to the construction of VUVCD spectrophotometers using SR as an intense light source: Brookhaven National Lab (Upton, NY), SRS (Daresbury, UK), ASTRID (Aarhus, Denmark), BESSY (Berlin, Germany), and BRSF (Beijing, China). New SR-VUVCD spectrophotometers are under construction at DIAMOND (UK), SOLEIL (Saint-Aubin, France), USTC (Hefei, China), LNLS (Campinas, Brazil), and Australian Synchrotron (Melbourne, Australia).

We started construction of BL15 and a VUVCD spectrophotometer using a small-scale SR source (0.7 GeV) at HiSOR in 1997. This system is capable of measuring the CD spectra of biomolecules in solutions from 310 to 140 nm by keeping all the optical devices under a high vacuum. We have applied this VUVCD spectophotometer for measuring the VUVCD spectra of various amino acids, saccharides, and proteins, and confirmed its usefulness for the structure analyses of these biomolecules in solution. Overview of BL15, VUVCD spectophotometer, performance, and future prospects are briefly described below.

BL15 is composed of four mirrors (M0, M1, M2, and M3) and a McPherson's Model 225M2 1-m normal incidence monochromator (NIM), which consists of two interchangeable concave gratings coated with Pt (1200 lines/mm) and MgF2/Al (1200 lines/mm) (Fig. 1). The NIM is available for the photon energy from 4 to 40 eV, which correspond to 310-31 nm. The resolution of the monochromator is 0.2 Ǻ.

Fig. 1. Overview of BL15.

The VUVCD spectrophotometer is set at the end-station of BL15. The spectrophotometer consists of two separate vacuum chambers, i.e., polarization modulation chamber and sample chamber (Figs. 2 and 3). The SR light monochromated by NIM is separated into two orthogonal linearly-polarized light beams by a Karl Lambrecht MgF2 Rochon prism (POL). Both linearly-polarized light beams are modulated to circularly polarized light beams at 50 kHz by a JASCO LiF photo-elastic modulator (PEM). In order to control PEM accuratelyand to stabilize the lock-in amplifier under a high vacuum, the optical servo-control system was used. The main light beam in the center of the chamber is led to the sample cell and the CD signal is detected with a Hamamatsu photomultiplier tube (PM). The other light beam is used as the reference signal to synchronize the polarization modulation.

Fig.2. Overview of VUVCD spectrophotometer.

Fig. 3. Block diagram of VUVCD spectrophotometer.

The optical cell consists of a stainless steel container with a cylindrical screw and two MgF2 windows of 20-mm diameter and 1-mm thick (Fig. 4). A c-axis-cut MgF2 disc is used to eliminate the birefringence of the windows. The optical cell is adjustable for the path length from 1.3 μm (without a spacer) to 50 μm using a doughnut-shaped Teflon spacer. The sample solution in the cell is sealed with three fluoride-rubber O-rings that can be pressed uniformly by the cylindrical screw. The temperature of the cell is controlled using a Peltier thermoelectric unit in the range from -30 to 80°C.

Fig. 4. Block diagram and overview of optical cell.

This spectrophotometer was first applied for measuring the VUVCD spectra of amino acids down to 140 nm in water (Chem. Lett. 2002). Optical cell with a temperature-control unit was developed (Anal. Sci. 2003). The VUVCD spectra of mono- and di-saccharides were measured down to 160 nm and the contributions of anomeric forms at C1 and gauche/trans conformation of hydroxymethyl group at C5 were estimated by a deconvolution analysis of the spectra (Carbohydr. Res. 2004). The VUVCD spectra of 31 proteins at native form were measured down to 160 nm, and the content and the segment number of secondary structures were successfully predicted (J. Biochem. 2004, 2005). The VUVCD spectrum of alanine was assigned using the time-dependent density functional theory (J. Phys. Chem. 2005). The VUVCD spectra of denatured proteins were measured down to 172 nm and the characteristic secondary structures at various denaturation states were clarified (Biophys. J. 2007). We have developed the method for predicting the positions of α-helix and β-strand on the amino-acid sequence by combining the VUVCD spectra with a neural network (in submission). The VUVCD analyses of amyloid proteins, S-S bond deleted proteins, alcohol denatured proteins are in progress in collaboration with some other groups. Details of these studies are shown in the Activity Reports attached.

These VUVCD studies have been elected the invited lecture in many international conferences: 8th International Conference on Circular Dichroism (2001), 3rd Singapore International Chemical Conference (2003), 8th International Conference on Biology and Synchrotron Radiation (2004), International Symposium on Circular Dichrosim Spectroscopy in Structural Molecular Biology (2005), 10th International Conference on Circular Dichroism (2005), 10th Hiroshima International Symposium on Synchrotron Radiation (2006). The poster presented at 8th International Conference on Circular Dichroism got a poster award. The paper published in Carbohydr. Res. (2004) was awarded to the "Top 50 most cited papers"of this journal as published in 2004-2007. These activities were also introduced as a topics in the Science Newspaper of March 26, 2004 (Japanese).

SR is an intense light source useful for VUVCD measurements, but the strong SR beam often damages biomolecules even on short-time irradiation. Therefore, the optimized photon flux and S/N ratio are desirable for measuring accurately the VUVCD spectrum. Fortunately, no damage of biomolecules has been so far observed in our VUVCD measurements because the SR power at BL15 is not so strong (1010 photon/sec). However, more powerful beam is desirable for further improvement of S/N ratio and shortening the measurement time of VUVCD spectra. Therefore, this VUVCD system is decided to be moved to BL12 which has a photon flux of 1012 photon/sec. At that chance, the normal incidence monochromator is planed to be changed from the McPherson type to the Wadsworth type which is more suitable for VUVVD measurements. Although BL12 is under construction, we expect that the increase in photon flux of 102 order would largely improve the performance of VUVCD spectrophotometer. A new VUVCD instrument with a multi-channel system is also considered to make the CD measurements more efficient. This system would also make possible the monitoring of dynamics or conformational change of proteins and DNA by utilizing the pulse nature of SR beam. A serious problem for continuing the VUVCD study is a lack of staff. At present, only one post-doctoral fellow engages in this study in collaboration with an emeritus professor. For future development of VUVCD spectroscopy at HiSOR, it is indispensable to promptly get hold of a good personnel with a steady position.

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