Supramolecular Complex Formation and Crystallization of Isocitrate Dehydrogenase fromThermus thermophilusHB8: 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 ¼ 495:5, b ¼ 189:2, c ¼ 336:2 Å , and ¼ 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.

Shuffling genes around in hot environments: the unique DNA transporter ofThermus thermophilus

“…The proteins from thermophiles exhibit unusually high stabilities and have a greater ability to crystallize compared with their mesophilic counterparts, which makes evident the relevance of the T. thermophilus DNA translocator as a model for further studies at the structural level. The crystallization potential of T. thermophilus proteins is supported by numerous reports of successful crystallization of T. thermophilus proteins and protein complexes, including V‐ATPase, isocitrate dehydrogenase, uroporphyrinogen II synthase, an outer membrane protein and 360 proteins of T. thermophilus HB8; the latter were successfully crystallized in a whole cell‐project (Toei et al , 2007; Brosig et al , 2008; Iino et al , 2008; Ishii et al , 2008; Schubert et al , 2008).…”

Crystallization, Structure and Functional Robustness of Isocitrate Dehydrogenases

“…Separately, we have performed AFM scanning on the crystalline surface of crystal form II. A lot of ellipsoidal bodies were observed (N. Ishii et al, 2008). The average values of the short and long axes of the ellipsoidal bodies detected in AFM imaging are 10.87 ± 1.47, 18.61 ± 2.58 nm, respectively.…”

“…Furthermore, the crystals in form II, having the same appearance, grown in the slightly basic pH region, pH 7.8, 8.1 and 8.4, for example, could never diffract X-rays at 95 K. These observations could be understood in that the formation of the supramolecular units and the interaction between the units were suitable enough to form the form II crystal shape at room temperature, which could be further inferred from the diffraction images in high resolution range (data not shown). When the crystals were treated at cryogenic condition, the intermolecular interactions should have been altered in the direction of increasing entropy (N. Ishii et al, 2008). Tth ICDH molecules were placed under the crystallization condition for about three months and the protein forming crystal form II were examined as they were at the state with HPLC gel filtration chromatography using a TSKgel G3000SWxl (Tosoh, Tokyo, Japan).…”

Folding and Binding Properties of Human Complement Receptor Type 1 Extracellular Domain

“…Crystallization using thus obtained human sCR1 was carried out with a normal sitting drop vapor diffusion technique in the combination with a random-screening protocol with screen packages such as the Hampton Crystal Screen, Hampton Crystal Screen II (Hampton Research, CA), and Structure Screen 1 and Structure Screen 2 (Molecular Dimensions, Suffolk, UK) after the condensation to the protein concentration of around 3.5 mg/mL by means of centrifugal concentrator (Centri-plus 20, Merck Millipore Corp., MA). The incubation temperature for the crystallization trials was kept at around 23°C [4,25].…”