**8. References**

302 Current Trends in X-Ray Crystallography

and krypton were bound within GBS II with a quite low occupation, and only xenon was bound within GBS I. Argon did not bind to lysozyme even at a pressure of 50 bar. The rule that showed that gas occupancy rose with gas size and polarizability is almost respected, since the small size of GBS II could prevent xenon binding and thus favour krypton binding. In the pressure range which would correspond to physiological conditions (5 – 10 bar), xenon occupancy is likely to be very low (less than 10 %), and krypton and argon occupancies are null. It is then likely than enzymatic activity of lysozyme is not modified by

Noble gases bind to proteins essentially through non-covalent van der Waals interactions, their binding constant depending on their electronic polarizability. In the three studied enzymes, gas occupancies were in the order of their polarizability, Xe > Kr > Ar, as it was already found for T4 lysozyme (Quillin et al., 2000). The only slight exception was the internal cavity of egg white lysozyme, where its smaller size prevented xenon higher

The major physiological targets of gaseous anesthetics are postulated to be neuronal channels receptors, like the NMDA receptor which is inhibited by xenon (Campagna et al., 2003; Franks, 2008), or the GABAA receptor which could be modulated by argon (Abraini et al., 2003). However, at lower concentration, gas would bind mainly to globular targets, amongst them enzymes, whose functions would be modulated by the presence of inert gas. From the present study, we can infer different mode of inhibition by gas. In urate oxidase, gas inhibited the catalytic reaction through an indirect mechanism; the presence of the gas within the cavity would prevent the cavity contraction, thus modifying the active site flexibility. In elastase, gas inhibited the catalytic reaction through a direct mechanism; the presence of the gas in the active site would prevent the substrate binding. In lysozyme, gas

Protein activity requires some conformational flexibility (Frauenfelder et al., 1991). In enzymes, the balance between conformational flexibility and rigidity is adjusted to optimize the catalytic efficiency for a given condition (Chiuri et al., 2009). Cavities would facilitate conformational changes and are though to play a key role in protein function (Hubbard et al., 1994). Anesthetics have been postulated to act by stabilizing high-energy conformers inducing altered functions (Eckenhoff, 2001; Johansson et al., 2005). High pressure was also postulated to stabilize high-energy conformers with altered functions (Frauenfelder et al., 1990; Akasaka, 2006; Fourme et al., 2006). Urate oxidase is thus a key example which highlights the effect of anesthetic, since this enzyme is both inhibited by gas presence in a

hydrophobic cavity (Marassio et al., 2011) and by high pressure (Girard et al., 2010).

modulated (inhibited or potentiated) by the presence of gas.

Inert gas binds to proteins through very weak non-covalent van der waals interaction. How such weak interactions could generate such high biological effect such as anesthesia ? It was suggested that anesthesia would arise from small effects at many biological targets (Eckenhoff, 2001). The present study would confirm this hypothesis, showing that some enzymes could be stabilized by the presence of gas in hydrophobic cavity (as in urate oxidase), some enzymes could be directly inhibited by gas (as in elastase case), and some enzymes are not affected by gas (as in the case of lysozyme). The mechanisms of neuroprotection and anesthesia by inert gases are thus very complex processes with many biological targets whose function are

would not inhibit the catalytic reaction, their occupation being too weak.

the presence of an inert gas.

**6. Conclusion** 

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**13** 

*Japan* 

Noriyuki Ishii

**Crystallization, Structure and Functional** 

*National Institute of Advanced Industrial Science and Technology (AIST)* 

**Robustness of Isocitrate Dehydrogenases** 

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

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

**1. Introduction** 

stage.

