New Structural Analysis Methods for Hemoglobin Crystals. Time-Resolved Cryogenic X-Ray Crystallography with Extended Pulsed-Laser Pumping and 3D Imaging by X-Ray Fluorescence Holography

Abstract

Hemoglobin (Hb) has long served as a model for studying protein allostery in general. Static crystal structures of deoxy tense (T) state and oxy relaxed (R) state of Hb reveal multiple differences at both tertiary and quaternary levels. However, we still lack an adequate understanding of how these structural differences control ligand binding. Here we present two methods for Hb crystals to investigate the ligation-linked protein motion and the atomic details of the heme active site structure.The first method is time-resolved X-ray crystallography combined with high-repetition pulsed-laser pumping and cryogenic trapping. At cryogenic temperatures under 50 K, a photo-dissociated ligand molecule can move to only the limited range near the heme, and the protein moiety can undergo only small structural changes. By contrast, at 100-140 K under continuous pulsed-laser irradiation, we found that ligand migration and protein relaxation can be observed. We have succeeded in tracking the photo-dissociated ligand molecule that migrates along the pathway connecting inner cavities of Hb in a different direction in each subunit.The second method is atomic-resolution X-ray fluorescence holography (XFH), which utilizes fluorescing atoms as a wave source within a crystal sample. So far, XFH has generally been applied to inorganic materials to investigate the microscopic environments of trace metals. This is the first application of XFH for protein crystals. For Hb crystals, XFH can provide the three dimensional atomic images around the iron central atoms of the individual hemes. We have carried out XFH measurements at 100 K of CO-bound Hb crystals at BL6C, KEK, Japan. We will present our latest data and share with you the results and discussion of this work.