X-Ray Fluorescence Holography for Proteins: Application to Hemoglobin and Myoglobin

Abstract

X-ray fluorescence holography (XFH) is a 3-dimensional visualization method of atomic structures around specific atoms in single crystals (Tegze, M., Faigel, G. (1996) Nature 380, 49). In XFH, X-ray fluorescence atoms (usually metal atoms) are excited by the external X-rays. The fluorescence intensity is spatially modulated to form the holograms, because the fluorescent waves are partially scattered by neighboring atoms and interfere with unscattered reference waves. Compared to standard X-ray diffraction methods, a remarkable feature of XFH is that it provides a means of extracting both intensity and phase information, allowing “model-free” structural analysis using Fourier-like transform of the observed hologram (Barton, J. J. (1991) Phys. Rev. Lett. 67, 3106). Here we apply XFH to protein crystals with the recently developed measurement system (Sato-Tomita, A. et al. (2016) Rev. Sci. Inst. 87, 063707). The model protein used in this study is hemoglobin (Hb), an (αβ)2 tetrameric protein with an iron-containing heme group per subunit. Since the Hb crystal has orientationally different multiple molecules in the unit cell, we used a pseudo symmetric-operation on each heme group to extract structural information of a particular molecule of interest. Using such an analysis, we obtained the atomic images of the heme environments in the individual subunit of Hb. The results indicate that the degree of planarity of the α heme is higher than that of the β heme, consistent with the result of high-resolution X-ray crystallography. In the conference, we will also present the results of a monomeric hemoprotein, myoglobin, with a similar folding as a Hb subunit, serving as a simpler model protein.