**1. Introduction**

308 Current Trends in X-Ray Crystallography

Vitali, J., Robbins, A.H., Almo, S.C. & Tilton, R.F. (1991). Using xenon as a heavy atom for

Yamakura, T. & Harris, R.A. (2000). Effects of gaseous anesthetics nitrous oxide and xenon

5: pp. 931-935.

Vol. 93 No. 4: pp. 1095-1101.

determining phases in sperm whale met-myoglobin. *J. Appl. Crystallogr.* Vol. 24 No.

on ligand-gated ion channels. Comparison with isoflurane and ethanol. *Anesthesiol.*

Understanding in detail the function of proteins and complexes requires knowledge of their three-dimensional structure. Historically, early development of protein crystallization, only provided a means for the purification of specific proteins from an impure mixture and an index that a protein had been purified (McPherson, 2004). X-ray diffraction analysis in combination with crystallization has become indispensable techniques for establishing the properties and nature of catalytic macromolecules. In spite of remarkable progress over the last two decades in the overexpression of macromolecules, in sophisticated screening of crystallization conditions, and X-ray data collection and analysis, the determination of novel structures by X-ray crystallography is still largely limited by the difficulty of obtaining highquality crystals of interest and maintaining their quality throughout the data collection stage.

The property of self-assembly exhibited by biological macromolecules plays a central role in biology. Most of the primary stage of the self-associated construction of supramolecular structures such as macromolecular complexes, assemblies, organelles, cell membranes, cytoskeletons, and so on, involves only the establishment of weak and oriented interactions between homologous molecules. The formation of protein crystals can be described as the expression of the self-assembly properties of the constituent molecules placed under favorable conditions. In order to engineer proteins that possess various kinds of physicochemical properties as well as biochemical functions within nano-assemblies, we need to understand features of the intermolecular interactions and the capacities for macromolecules to self-associate that govern the integrating of protein assemblies, such as seen in the primary stage of crystallization and crystal growth processes of protein molecules (Akiba et al., 2005; N. Ishii et al., 2001). It further requires information to specify how the building block molecules are joined into higher order structures. The fundamental and practical importance of these processes motivates the interest of studying self-assembly (self-organization). Crystals of a certain kind of protein belonging to different space groups may provide a better understanding of intermolecular interactions which can guide the development of techniques to manipulate the orientation of each protein molecule and arrangements in the construction of nano-architectures using the desired protein. The conformation of protein molecules as well as configurations of amino-acid residues are often stabilized in the supramolecular complex through the cumulative effects of various

Crystallization, Structure and Functional Robustness of Isocitrate Dehydrogenases 311

concentration of the sample protein, ligands, inhibitors, genetic or chemical modifications,

*Crystallization conditions*

Concentration of the proteins, Ligands, Inhibitors, Effectors, …

Fig. 1. Conceptual diagram of crystallization condition search screening on the landscape of potential free energy. Various parameters are shaken from certain starting conditions.

Precipitant type, concentration, pH, Temperature, Ionic strength …

In our crystallization study of ICDH from a thermophile, *Thermus thermophilus* HB8 (*Tth* ICDH), *Tth* ICDH was overexpressed in *E. coli* MV1190 which carried plasmids pKID1, and the gene product was purified according to reported methods (Miyazaki et al., 1994). Purity of the yielded protein was checked with the polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) (Laemmli, 1970). After dialysis against pure water, the protein was stored at 277 K until use. The crystallization experiments were carried out by the hanging drop vapor diffusion method in a 24-well tissue-culture linbro plate (Iwaki Glass Co., Ltd., Ciba, Japan) at 298 and/or 277 K. A random-screening protocol with screens developed in-house was used. The initial hits were optimized with further finer grid search. One screen package is similar to the Hampton Crystal Screen and Crystal Screen II (Hampton Research, Aliso Viejo, CA), and the other screen package contains various additives such as cofactors, inhibitors, nucleotides, minerals, salts and buffers with pH range 4 - 9. *Tth* ICDH was dialyzed against 20 mM Tris-HCl, pH 7.2, at 277 K, and incubated at 333 K for 10 min before the crystallization experiments. The initial protein solution contained 10.2 mg/ml of *Tth* ICDH in 20 mM Tris-HCl, pH 7.2. To a droplet of the protein solution, an equal amount of reservoir solution was added and then the droplet was equilibrated over 0.5 ml reservoir solution. The resulting microcrystals were obtained at the conditions with the reservoir commonly containing 100 mM sodium cacodylate and 1.4 M sodium acetate. The crystallization conditions were further optimized with regards to pH, co-existence of DL-isocitric acid, citric acid and/or cations like Mg2+ and Mn2+. During the survey for crystallization conditions, information on protein crystallization such as

and so on.

*Free energy*

intermolecular interactions, such as salt bridges and hydrogen bonds, on the surface. Even in monomeric or dimeric proteins under physiological conditions sometimes seen as highly oligomeric complexes during crystal structure determination.

### **2. Isocitrate dehydrogenase**

Isocitrate dehydrogenase (ICDH, EC 1.1.1.42) is a metal dependent (Mg2+ or Mn2+) enzyme that plays an important role in the tricarboxylic acid cycle. It lies at a critical juncture between the cycle and the glyoxylate pathway to the biosynthesis of glutamate. The enzyme catalyzes the subsequent oxidative decarboxylation reaction of 2R,3S-isocitrate to yield 2 oxoglutarate and carbon dioxide with the protonation of NAD or NADP in the cycle. The 2 oxoglutarate is known to be a key substrate in the biosyntheses of cell constituents via reductive amination to glutamate. These pathways are among the first to have evolved in the history of life (Melendez-Hevia et al., 1996). The ICDHs have been distinguished into three subfamilies based on sequence comparisons (Steen et al., 1997, 2001). All of the archaeal and most of the bacterial ICDHs are classified together into subfamily I, eukaryotic homodimeric ICDHs and some bacterial ICDHs are categorized as subfamily II, eukaryotic hetero oligomeric ICDHs constitute subfamily III. In contrast to these homologous proteins, another type of NADP+-dependent monomeric ICDHs with molecular mass of 80-100 kDa have been found (Chen & Gadal, 1990). The active site of these enzymes in this category must be constructed from the side chains of residues within a single polypeptide chain. Although the monomeric ICDH catalyzes a reaction identical to that of the dimeric ICDH, no homology in the primary sequence has been found between the monomeric and dimeric ICDHs (Sahara et al., 2002). In addition, immunological studies suggest that monomeric and dimeric ICDHs are not structurally homologous (Fukunaga et al., 1992; Leyland & Kelly, 1991). A certain bacterium such as *Calwellia maris* possesses both monomeric and dimeric ICDHs (Yoneta et al., 2004). It seems that transcription of both genes is regulated in response to environmental factors.
