**5.1 Introduction on lysozyme structures under inert gas pressure**

Egg white lysozyme C (EC 3.2.1.17) is an enzyme of 129 residues which hydrolyse peptidoglycans. It crystallizes in tetragonal space group P43212 with one monomeric enzyme per asymmetric unit (cell: a = 79.2 Å, b = 79.2 Å, c = 37.9 Å, = = = 90°). In the different crystallographic structures of lysozyme in presence of xenon deposited in the Protein Data Bank : 1C10 (Prangé et al., 1998), 2A7D (Mueller-Dieckmann et al., 2004) and 1VAU (Takeda et al., 2004), xenon was bound mainly in a pocket at a crystallographic interface (gas binding site I or GBS I in Figure 8) and weakly in a small internal cavity (GBS II in Figure 8). In the native gas-less structure, a water molecule was present in GBS I, and GBS II was empty. GBS I is moderately hydrophobic, lined with 55 % carbons, and GBS II is very hydrophobic, lined with 83 % carbons. The occupancy of xenon ranged between 30 and 60% in GBS I and stayed beyond 25 % in GBS II.

Fig. 8. Xenon binding sites in lysozyme, shown with two symmetrical subunits. A. Xenon was bound at the crystallographic interface between two symmetrical monomers (GBS I) and a second xenon was bound within a small internal cavity buried within each monomer (GBS II) (xenon shown as orange spheres, and lysozyme with its C chain as ribbon, one symmetric is colored in green, the second in blue). B. Solvent-accessible surface of two symmetrical lysozymes.

In the structure 1C10, a third xenon was located in the active site, where either two water molecules or one water molecule and one chloride ion were found in the other deposited structures. In the structure 1VAU, a third xenon was located in another crystallographic pocket at the interface between two monomers, where a water molecule was found in the other deposited structures.

In the crystallographic structure of lysozyme under a 55 bar pressure of krypton 1QTK, (Schiltz et al., 1997; Prangé et al., 1998), krypton was bound in the internal cavity (GBS II) but not in the crystallographic pocket (GBS I).

In the present study, the structure of a native gas-less lysozyme was determined in the same condition than structures under inert gas pressure, i.e. at room temperature in quartz capillary. Two native structures have been solved, at 1.6 Å and 1.55 Å resolution. In both structures, the internal cavity GBS II was empty and there was a water molecule in GBS I which had a B-

Protein-Noble Gas Interactions Investigated by Crystallography

Krypton pressure (bar)

confirm this prediction.

map in GBS II.

on Three Enzymes - Implication on Anesthesia and Neuroprotection Mechanisms 301

GBS I (%)

5 1.55 0 0 12.1 10 1.60 0 0 4.6 20 1.55 0 10 14.9 55 (1QTK) 2.03 0 49 7.1 Table 9. Krypton pressure, resolution of the crystallographic structures, krypton occupancy

A theoretical study based on a three-dimensional distribution function theory of molecular liquids was applied to lysozyme in presence of water and noble gases (Imai et al., 2007). This study predicted that krypton had a slightly better affinity for GBS II than xenon. Structures of lysozyme under higher pressure of krypton (30 and 40 bar) would be necessary to

0 10 20 30 40 50 60 **Pressure (bar)**

**Xe** 

The structures of lysozyme under argon pressure of 10 to 50 bar were determined in the present study. Whatever the applied pressure, no argon was visible in the electron density map even at a pressure of 50 bar neither in GBS I or in GBS II. In GBS I, the water molecule was present at the exact place of the water molecule in the native gas-less structure. The smaller size of argon should not prevent argon to bind within GBS II, however, a higher pressure would probably be necessary to be able to visualize argon in the electron density

In the theoretical study already mentioned (Imai et al., 2007), argon affinity for GBS II was predicted to be very high. However, the present study showed that even at high pressure, argon did not bind within GBS II, even if it smaller size would allow its binding. Argon polarizability is lower than krypton and xenon polarizabilities (Table 1), due to its small

Lysozyme possesses two gas binding sites, one located in a pocket at a crystallographic interface (GBS I) and one located within a quite small internal cavity (GBS II). Both xenon

number of electron, and could explain its very low affinity for lysozyme.

**5.5 Conclusion on lysozyme structures under inert gas pressure** 

Kr occ. in GBS II (%)

**( )**

Kr-induced expansion of GBS II (%)

Resolution (Å) Kr occ. in

in GBS I , krypton occupancy in GBS II and krypton-induced expansion of GBS II.

**Kr**

Fig. 9. Xenon and krypton occupancies as a function of pressure.

**5.4 Structure of lysozyme under pressure of argon** 

**Occupancy (%)**

factor between 25 and 29 Å2. The volume of the internal cavity GBS II is very small, about 40 Å3 while the volume of GBS I at a crystallographic interface is larger, around 100 Å3.

#### **5.2 Structure of lysozyme under pressure of xenon**

The structures of lysozyme under xenon pressure of 10 to 30 bar were determined in the present study. Xenon was bound in GBS I with an occupancy which did not increase with pressure and remained around 20 and 30 % (which is the double of the occupancy for the monomer since the xenon is located at a crystallographic interface). Xenon was also bound in GBS II with an occupancy which increased with the applied pressure but which remained quite low (20 % occupancy at 30 bar).

Xenon occupancy in GBS I remained stable whatever the applied pressure and induced almost no expansion of its volume (less than 10 %). Xenon location was not exactly superposed to the water molecule location present in the native gas-less structure, with a distance of 0.6 to 0.7 Å between them. This gas binding site being located at a crystallographic interface is likely to be not physiological.


Table 8. Xenon pressure, resolution of the crystallographic structures, xenon occupancy in GBS I , xenon occupancy in GBS II and xenon-induced expansion of GBS II.

Xenon occupancy in GBS II remained very low, due to the small size of the cavity (volume around 40 Å3). Xenon van der Waals radius is of 2.21 Å, its theoretical volume is thus of 45 Å3, close to the volume of GBS II. Xenon induced an expansion of the volume of this small internal cavity which reached a volume around 50 Å3 for xenon occupancy of 20 %. In the range of pressure corresponding to physiological conditions (5 – 10 bar), the occupation of xenon in GBS II is likely to be very small (less than 10 %).

#### **5.3 Structure of lysozyme under pressure of krypton**

The structures of lysozyme under krypton pressure of 5 to 20 bar were determined in the present study. No krypton was visible in GBS I whatever the applied pressure, as it was the case in the structure determined under a krypton pressure of 55 bar. In all cases, there was a water molecule at the exact same location than in the native gas-less structure. Krypton was bound weakly in GBS-II and became visible in the electron density map at a pressure of 20 bar. Krypton induced an expansion of the volume of GBS II which seemed not related to krypton occupancy. However, this krypton-induced expansion of GBS II indicated that krypton was present event when it was not visible in the electron density map (Table 9).

Krypton is smaller than xenon (van der Waals radius of 2.03 Å instead of 2.21 Å, and volume of 35 Å3 instead of 45 Å3), it is thus likely than krypton occupancy can reach higher value than xenon occupancy in GBS II (Figure 9).

factor between 25 and 29 Å2. The volume of the internal cavity GBS II is very small, about 40

The structures of lysozyme under xenon pressure of 10 to 30 bar were determined in the present study. Xenon was bound in GBS I with an occupancy which did not increase with pressure and remained around 20 and 30 % (which is the double of the occupancy for the monomer since the xenon is located at a crystallographic interface). Xenon was also bound in GBS II with an occupancy which increased with the applied pressure but which remained

Xenon occupancy in GBS I remained stable whatever the applied pressure and induced almost no expansion of its volume (less than 10 %). Xenon location was not exactly superposed to the water molecule location present in the native gas-less structure, with a distance of 0.6 to 0.7 Å between them. This gas binding site being located at a

GBS I (%)

10 1.55 30 10 9.1 15 1.60 20 10 4.6 20 1.55 30 12 6.9 25 1.90 24 15 13.6 30 1.65 30 20 23.4 Table 8. Xenon pressure, resolution of the crystallographic structures, xenon occupancy in

Xenon occupancy in GBS II remained very low, due to the small size of the cavity (volume around 40 Å3). Xenon van der Waals radius is of 2.21 Å, its theoretical volume is thus of 45 Å3, close to the volume of GBS II. Xenon induced an expansion of the volume of this small internal cavity which reached a volume around 50 Å3 for xenon occupancy of 20 %. In the range of pressure corresponding to physiological conditions (5 – 10 bar), the occupation of

The structures of lysozyme under krypton pressure of 5 to 20 bar were determined in the present study. No krypton was visible in GBS I whatever the applied pressure, as it was the case in the structure determined under a krypton pressure of 55 bar. In all cases, there was a water molecule at the exact same location than in the native gas-less structure. Krypton was bound weakly in GBS-II and became visible in the electron density map at a pressure of 20 bar. Krypton induced an expansion of the volume of GBS II which seemed not related to krypton occupancy. However, this krypton-induced expansion of GBS II indicated that krypton was

Krypton is smaller than xenon (van der Waals radius of 2.03 Å instead of 2.21 Å, and volume of 35 Å3 instead of 45 Å3), it is thus likely than krypton occupancy can reach higher

Xe occ. in GBS II (%)

Xe-induced expansion of GBS II (%)

Å3 while the volume of GBS I at a crystallographic interface is larger, around 100 Å3.

**5.2 Structure of lysozyme under pressure of xenon** 

crystallographic interface is likely to be not physiological.

xenon in GBS II is likely to be very small (less than 10 %).

**5.3 Structure of lysozyme under pressure of krypton** 

value than xenon occupancy in GBS II (Figure 9).

Resolution (Å) Xe occ. in

GBS I , xenon occupancy in GBS II and xenon-induced expansion of GBS II.

present event when it was not visible in the electron density map (Table 9).

quite low (20 % occupancy at 30 bar).

Xenon pressure (bar)



Fig. 9. Xenon and krypton occupancies as a function of pressure.

A theoretical study based on a three-dimensional distribution function theory of molecular liquids was applied to lysozyme in presence of water and noble gases (Imai et al., 2007). This study predicted that krypton had a slightly better affinity for GBS II than xenon. Structures of lysozyme under higher pressure of krypton (30 and 40 bar) would be necessary to confirm this prediction.

## **5.4 Structure of lysozyme under pressure of argon**

The structures of lysozyme under argon pressure of 10 to 50 bar were determined in the present study. Whatever the applied pressure, no argon was visible in the electron density map even at a pressure of 50 bar neither in GBS I or in GBS II. In GBS I, the water molecule was present at the exact place of the water molecule in the native gas-less structure. The smaller size of argon should not prevent argon to bind within GBS II, however, a higher pressure would probably be necessary to be able to visualize argon in the electron density map in GBS II.

In the theoretical study already mentioned (Imai et al., 2007), argon affinity for GBS II was predicted to be very high. However, the present study showed that even at high pressure, argon did not bind within GBS II, even if it smaller size would allow its binding. Argon polarizability is lower than krypton and xenon polarizabilities (Table 1), due to its small number of electron, and could explain its very low affinity for lysozyme.

#### **5.5 Conclusion on lysozyme structures under inert gas pressure**

Lysozyme possesses two gas binding sites, one located in a pocket at a crystallographic interface (GBS I) and one located within a quite small internal cavity (GBS II). Both xenon

Protein-Noble Gas Interactions Investigated by Crystallography

**7. Acknowledgement** 

work.

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on Three Enzymes - Implication on Anesthesia and Neuroprotection Mechanisms 303

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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 the presence of an inert gas.
