3.3.2. Halophiles

Halophiles grow in the presence of high concentrations of salts. In order to avoid osmotic shock, they accumulate inorganic salts or small organic molecules in the cytoplasm until the intracellular osmolarity equals the extracellular ion concentration [101]. They also require their proteins to operate under extreme ionic conditions. Actually, halophilic enzymes require high salt concentrations for activity and stability (1–4 M range) [104]. Stabilization of proteins in high salinity environments is linked to the interaction of hydrated ions with negatively charged surface residues. A highly ordered shell of water molecules is formed that protects the protein and prevents denaturation [105]. Compared to non-halophilic proteins, halophilic enzymes present less non-polar residues and more charged residues on the protein surface, a higher frequency of acidic (Asp and Glu) over basic residues (Lys), and low hydrophobicity [105] (Figure 6). This high surface charge is neutralized mainly by tightly bound water dipoles [22]. Such excess of acidic over basic amino acid residues makes them more flexible at high salt concentrations in conditions where non-halophilic proteins tend to aggregate [106,107]. Halophilic proteins seem to be specially adapted to have multiple crossover adaptations, especially to pH and temperature.

**Figure 6.** Coulombic surface maps for both sides of the *Salinibacter ruber* (Sr), *Haloarcula morimortui* (Hm), *Haloferax volcanii* (Hv) and *Methylobacterium extorquens* (Me) malate dehydrogenases. The halophilic structures display negative surface areas (in red), a common feature of halophilic proteins. Reprinted with permission from [101].
