Supramolecular Complex Formation and Crystallization of Isocitrate Dehydrogenase from Thermus thermophilus HB8: Preliminary Studies with X-Ray Crystallography and Atomic Force Microscopy

Abstract

Atomic force microscopy (AFM) observation of a crystal surface of the thermostable isocitrate dehydrogenase (ICDH) from a thermophilic eubacterium, Thermus thermophilus HB8, suggested that the crystal consists of huge homo-complexed ellipsoidal bodies of the protein, with averaged long- and short-axis diameters of 18.6 nm and 10.9 nm respectively. Thick diamond-shaped crystals of about 0.4 mm on the longest axis were obtained by the vapor diffusion method from a solution of 100 mM sodium cacodylate, pH 6.6–8.4, containing 1.4 M sodium acetate as the precipitate, and diffracted X-rays at 3.7 Å resolution. The crystals belonged to the monoclinic lattice type with space group C2 and had cell dimensions of a=495.5, b=189.2, c=336.2 Å, and β=126.4°, indicating that an asymmetric unit contained more than 33 molecules with a molecular mass of 54.2 kDa. Calculations based on data obtained by the X-ray method showed good agreement with AFM observation. These results suggest the possibility that the residing T. thermophilus HB8 ICDH molecules are piled one on top another as a preformed supramolecular nano-architecture in the crystal lattice. The system appears suitable for further investigation using a bottom-up approach to the self-associated construction of nano-architectures with proteins.

• atomic force microscopy
• crystallization
• nanostructure
• X-ray diffraction
• isocitrate dehydrogenase