Circular dichroism (CD) spectroscopy in the ultraviolet (UV) region is widely used for the secondary structural analysis of proteins in an aqueous solution. Although the structural information from CD spectra is limited compared with that from X-ray crystallography and nuclear magnetic resonance (NMR), both of which display three-dimensional structures with atomic-level resolutions, CD spectroscopy is a powerful tool because it can more easily provide structural information, including the structural dynamics, because of some notable advantages: (i) the required sample amount is smaller (1–10% of those needed for X-ray crystallography and NMR [1
]) and (ii) the samples can be easily prepared by simply dissolving the proteins in a solvent. Neither crystallization nor isotopic substitution is required. Therefore, the loss of the samples and accidental denaturations during sample preparation are negligible in most cases.
The use of synchrotron radiation (SR) as a light source for CD spectroscopy allows more precise structural information to be obtained, because an SR beam can expand the measurement region to the vacuum-ultraviolet (VUV) region where additional CD peaks are often observed. Indeed, synchrotron radiation circular dichroism (SRCD) spectroscopy has produced successful outcomes over the past two decades [2
]. It has also become desirable to reduce the sample volume with the increasing interest in scarce proteins that are difficult to synthesize.
Although the sample volume could be reduced using small-capacity cuvettes, this is insufficient in most cases of CD spectroscopy. Most commercial and SR-based CD spectrophotometers adopt a so-called “transmission method”, i.e., they detect transmitted light passed through the sample and provide CD spectra. In this case, the required sample volume largely depends on the size of the incident beam because the sample area must be larger than the beam size for the whole beam to pass through the sample. Therefore, most CD spectrophotometers require beam-size demagnification and cuvette capacity reduction.
One of the better ways to demagnify the beam size is using lenses and/or mirrors to focus the beam. Indeed, some groups have succeeded in measuring CD spectra using focal beams (although their motivations were different from that of this study). For example, Kane et al. focused the beam on a spot size of 20 × 60 μm2
using a focal lens and succeeded in measuring the CD spectra of a cytochrome c
protein using a micro flow channel of 200–250 nm at the German SR facility BESSY II [3
]. Yamada et al. developed a circularly polarizing microscope using the Schwarzschild objective (SO), combined with a convex mirror and polarizing undulator, at the Japanese SR facility TERAS. They achieved a sub-micron beam (0.66 μm at wavelength 200 nm) and obtained a CD image of a d
-10-camphorsulfonic acid film on a copper grid [4
]. For CD spectroscopy, the focusing systems must conserve the polarization degree, and chromatic aberrations must be eliminated in the wide wavelength range. The use of focal lenses is generally not enough to eliminate aberrations for wide-range CD measurements in the UV region to the VUV region. However, according to Yamada’s complicated system, it seems that the chromatic aberration due to the SO systems is negligible or small. Thus, for this study, the SO was installed on a VUV-CD spectrophotometer, which uses the SR light of beamline BL-12 [5
] of the Hiroshima Synchrotron Radiation Center (HiSOR), to demagnify the incident beam size. Small-capacity sample cells were also developed. As a result, the sample volume for CD spectroscopy was successfully reduced using the SO and cells. In addition, a CD spectrum of a scarce protein, lysine-36 trimethylated histone H3 protein (H3K36me3), was measured using the same system, and its secondary structural contents were analyzed.
In the next section, the performance of the new VUV-CD spectrophotometer measurement system and the application of this system to the structural analysis of H3K36me3 are reported.