**2. Determination of crystallographic structures of proteins under inert gases pressure**

To investigate the mechanism of interaction of gases with proteins, a structural approach using protein crystallography under gas pressure was developed. Xenon binds reversibly to proteins through non-covalent, weak energy van der Waals forces (Ewing et al., 1970). The first structures of protein – xenon complexes were solved in 1965 with myoglobin and haemoglobin under a xenon pressure of 2.5 bar, evidencing a xenon binding site in these two globins (Schoenborn, 1965; Schoenborn et al., 1965). At a pressure of 7 bar, four xenon binding sites were found in myoglobin indicating that the number of xenon binding sites rises with pressure (Tilton et al., 1984).

Since then, many structures of protein-xenon complexes were solved, with xenon used as a heavy atom in isomorphous replacement phasing method (MIR), because xenon has a high

Protein-Noble Gas Interactions Investigated by Crystallography

compared to physiological conditions.

W.L., DeLano Scientific, Palo Alto, CA, USA).

**comparison with** *in-vivo* **pharmacology effects** 

gas occupancy and hence with gas pressure.

**3. Structure of urate oxidase under inert gas pressure** 

**3.1 Structure of urate oxidase under pressure of xenon and nitrous oxide and** 

*Aspergillus flavus* urate oxidase (EC 1.7.3.3) is a homotetrameric enzyme of 301 residues per subunit which is involved in the oxidation of uric acid in presence of molecular oxygen. It crystallizes in the orthorhombic space group I222 with one monomer per asymmetric unit (cell: a = 79.8 Å, b = 96.2 Å, c = 105.4 Å, = = = 90°). X-ray structures of urate oxidase under various pressures of xenon and nitrous oxide have been determined. Both gases were bound mainly in an internal cavity close to the active site of the enzyme, this cavity being empty in the native gas-less structure (Figure 2). This cavity, completely buried within the monomer, is highly hydrophobic, with 86 % of the atoms lining the cavity being carbons. Both gases were bound also very weakly to a second location, a small extension of a solvent-accessible pocket quite hydrophobic (lined by 75 % carbons). The gas occupancy in this second binding site remained very low (less than 30 % at 30 bar of pressure). Gas occupancies in the main binding site were high, reaching saturation at 100 % for xenon and 60 % for nitrous oxide (Table 2). The main effect of the gas was to expand the volume of the cavity where it binds. This expansion increased with

at a crystallographic interface (Schiltz et al., 1997; Prangé et al., 1998).

on Three Enzymes - Implication on Anesthesia and Neuroprotection Mechanisms 289

xenon binds primarily in a large buried hydrophobic cavity close to the active site (Colloc'h et al., 2007; Marassio et al., 2011). Xenon was used as an isomorphous derivative during the determination of urate oxidase structure (Colloc'h et al., 1997). In elastase, like in most of the serine proteases, xenon binds within the specificity pocket S1 of the active site (Schiltz et al., 1995). In lysozyme, xenon binds weakly in an internal cavity and mainly in a pocket located

One of the drawbacks of using X-ray crystallography is the requirement to have a high gas pressure to be able to observe it in the electron density map. A gas pressure about 5 to 10 fold the physiological concentration is estimated to correspond to physiological conditions (Miller, 2002). In the present study, gas pressure ranges from 1 to 40 bar in order to reach a maximum occupancy at saturation, however, only the data between 5 and 10 bar can be

In the present study, diffraction data were collected at room temperature at the BM14, BM16 and BM30A beamlines at the European Synchrotron Radiation Facility (Grenoble, France). Detectors used were a MAR CCD detector for BM14, an ADSC Q210r CCD detector for BM16 and an ADSC Q315r CCD detector for BM30A. Data were indexed and integrated by *DENZO* and scaled independently and reduced using *SCALEPACK*, both programs from the *HKL* package (Otwinowski et al., 1997) or indexed and integrated by *MOSFLM* (Leslie, 2006) or *XDS* (Kabsch, 2010) and scaled by *SCALA*; intensities were converted in structure factor amplitudes and put on absolute scale using *TRUNCATE* and structure refinements were carried out by *REFMAC* (Murshudov et al., 1997), all programs from the *CCP4* package (Collaborative Computational Project, 1994). The graphics program *COOT* (Emsley et al., 2004) was used to visualize *|2Fobs – Fcalc|* and *|Fobs – Fcalc|* electron density maps and for manual rebuilding. Cavity volume were calculated with the program *CastP* (Dundas et al., 2006) with a probe radius of 1.3 Å. Structural figures were prepared using *PyMol* (deLano

number of electrons (54 e-) and binds with very little perturbation of the protein structure (Vitali et al., 1991; Schiltz et al., 1994; Bourguet et al., 1995; Colloc'h et al., 1997). On the other hand, krypton, though lighter than xenon, was popularized as an internal reference in anomalous phasing techniques MAD or SAD (Schiltz et al., 1997; Cohen et al., 2001) thanks to its absoption K edge at a convenient and useful wavelength easy to tune at all synchrotron places; for a review, see (Schiltz et al., 2003).

Xenon and other noble gas binds primarily in pre-existing hydrophobic cavities or pockets, very often empty in the native gas-less structures (Prangé et al., 1998). Xenon diffusing through protein atoms reaches easily its completely buried binding sites. Xenon was also used as an oxygen probe, based on the hypothesis that xenon and dioxygen would have equivalent binding sites (Duff et al., 2004). The comparison of the binding mode of xenon, krypton and argon was done on the phage T4 lysozyme, showing that gas occupancy rises with gas size and polarizability (Xe > Kr > Ar) (Quillin et al., 2000).

X-ray diffraction data of a protein under xenon pressure are collected either at liquid nitrogen temperature (100 K) or at room temperature. In the first case, the crystal inserted in a cryo-loop is placed in a xenon pressure chamber for a given time, then immediately after frozen in liquid nitrogen, to minimize the amount of xenon which could escape the protein crystal. The determination of the gas pressure within the crystal is thus quite imprecise. For the present study which needs the determination of protein structures under a large range of gas pressures, we have used a pressurisation cell in capillary, designed and developed for the preparation of isomorphous xenon derivatives (Schiltz et al., 1994; Schiltz et al., 2003).

Fig. 1. The pressurisation cell setting. A. Connection between the five elements shown in B. 1- Xenon bottle, 2- Gas regulator, 3- High precision gauge, 4- Bleeding valve, 5- Pressurisation cell.

Typically, a crystal of protein is placed inside a quartz capillary mounted on the pressurisation cell. The pressurisation cell is fixed on a standard goniometer head, and connected to a gas bottle. The pressure within the cell is determined precisely with a calibrated Ashcroft precision gauge (Figure 1). The pressure is maintained constant during all the data collection.

For the present study, we have investigated three different enzymes, urate oxidase, elastase and lysozyme in complex with three gases, xenon, krypton and argon. In urate oxidase,

number of electrons (54 e-) and binds with very little perturbation of the protein structure (Vitali et al., 1991; Schiltz et al., 1994; Bourguet et al., 1995; Colloc'h et al., 1997). On the other hand, krypton, though lighter than xenon, was popularized as an internal reference in anomalous phasing techniques MAD or SAD (Schiltz et al., 1997; Cohen et al., 2001) thanks to its absoption K edge at a convenient and useful wavelength easy to tune at all

Xenon and other noble gas binds primarily in pre-existing hydrophobic cavities or pockets, very often empty in the native gas-less structures (Prangé et al., 1998). Xenon diffusing through protein atoms reaches easily its completely buried binding sites. Xenon was also used as an oxygen probe, based on the hypothesis that xenon and dioxygen would have equivalent binding sites (Duff et al., 2004). The comparison of the binding mode of xenon, krypton and argon was done on the phage T4 lysozyme, showing that gas occupancy rises

X-ray diffraction data of a protein under xenon pressure are collected either at liquid nitrogen temperature (100 K) or at room temperature. In the first case, the crystal inserted in a cryo-loop is placed in a xenon pressure chamber for a given time, then immediately after frozen in liquid nitrogen, to minimize the amount of xenon which could escape the protein crystal. The determination of the gas pressure within the crystal is thus quite imprecise. For the present study which needs the determination of protein structures under a large range of gas pressures, we have used a pressurisation cell in capillary, designed and developed for the preparation of isomorphous xenon derivatives (Schiltz et al., 1994; Schiltz et al., 2003).

Fig. 1. The pressurisation cell setting. A. Connection between the five elements shown in B.

Typically, a crystal of protein is placed inside a quartz capillary mounted on the pressurisation cell. The pressurisation cell is fixed on a standard goniometer head, and connected to a gas bottle. The pressure within the cell is determined precisely with a calibrated Ashcroft precision gauge (Figure 1). The pressure is maintained constant during

For the present study, we have investigated three different enzymes, urate oxidase, elastase and lysozyme in complex with three gases, xenon, krypton and argon. In urate oxidase,

1- Xenon bottle, 2- Gas regulator, 3- High precision gauge, 4- Bleeding valve, 5-

Pressurisation cell.

all the data collection.

synchrotron places; for a review, see (Schiltz et al., 2003).

with gas size and polarizability (Xe > Kr > Ar) (Quillin et al., 2000).

xenon binds primarily in a large buried hydrophobic cavity close to the active site (Colloc'h et al., 2007; Marassio et al., 2011). Xenon was used as an isomorphous derivative during the determination of urate oxidase structure (Colloc'h et al., 1997). In elastase, like in most of the serine proteases, xenon binds within the specificity pocket S1 of the active site (Schiltz et al., 1995). In lysozyme, xenon binds weakly in an internal cavity and mainly in a pocket located at a crystallographic interface (Schiltz et al., 1997; Prangé et al., 1998).

One of the drawbacks of using X-ray crystallography is the requirement to have a high gas pressure to be able to observe it in the electron density map. A gas pressure about 5 to 10 fold the physiological concentration is estimated to correspond to physiological conditions (Miller, 2002). In the present study, gas pressure ranges from 1 to 40 bar in order to reach a maximum occupancy at saturation, however, only the data between 5 and 10 bar can be compared to physiological conditions.

In the present study, diffraction data were collected at room temperature at the BM14, BM16 and BM30A beamlines at the European Synchrotron Radiation Facility (Grenoble, France). Detectors used were a MAR CCD detector for BM14, an ADSC Q210r CCD detector for BM16 and an ADSC Q315r CCD detector for BM30A. Data were indexed and integrated by *DENZO* and scaled independently and reduced using *SCALEPACK*, both programs from the *HKL* package (Otwinowski et al., 1997) or indexed and integrated by *MOSFLM* (Leslie, 2006) or *XDS* (Kabsch, 2010) and scaled by *SCALA*; intensities were converted in structure factor amplitudes and put on absolute scale using *TRUNCATE* and structure refinements were carried out by *REFMAC* (Murshudov et al., 1997), all programs from the *CCP4* package (Collaborative Computational Project, 1994). The graphics program *COOT* (Emsley et al., 2004) was used to visualize *|2Fobs – Fcalc|* and *|Fobs – Fcalc|* electron density maps and for manual rebuilding. Cavity volume were calculated with the program *CastP* (Dundas et al., 2006) with a probe radius of 1.3 Å. Structural figures were prepared using *PyMol* (deLano W.L., DeLano Scientific, Palo Alto, CA, USA).
