Large-scale crystallization and neutron crystallographic analysis of HSP70 in complex with ADP

Abstract

HSP70 belongs to the heat-shock protein family and binds to unfolded proteins, driven by ATP hydrolysis, in order to prevent aggregation. Previous X-ray crystallographic analyses of HSP70 have shown that HSP70 binds to ADP with internal water molecules. In order to elucidate the role of the water molecules, including their H/D atoms, a neutron diffraction study of the human HSP70 ATPase domain was initiated. Deuterated large crystals of the HSP-ADP complex (1.2-1.8 mm³) were successfully grown by large-scale crystallization, and a neutron diffraction experiment at BIODIFF resulted in diffraction to a maximum resolution of 2.2 Å. After data reduction, the overall completeness, R_meas and average I/σ(I) were 90.4%, 11.7% and 8.1, respectively, indicating that the data set was sufficient to visualize H and D atoms.

Keywords: HSP70; heat-shock proteins; large-scale crystallization; neutron protein crystallography.

Figure 1

Crystal photographs of crystal 1 (a) and crystal 2 (b). The scale bars represent 1 mm.

Figure 2

Neutron diffraction patterns of crystal 1 (a) and crystal 2 (b) measured at BIODIFF at a wavelength of 3.4 Å.