Introduction and literature review
1.8 Salient features deduced from engineered extreme-stable mutants to better understand extreme-stability of proteins
1.8 Salient features deduced from engineered extreme-stable mutants to better
Table 1.5: Engineered mutants of extreme-stable proteins.
Mutated
protein Organism
No. of Mutants
in PDB
Method Property of mutant Region of
mutation Reason Thermostabilizing
Lysozyme Bacteriophage
T4 104
Directed evolution, site-directed mutagenesis
Enhanced thermostability
Loop, helix, strand
Enhanced core repacking,
aromatic interactions Psychrostabilizing
Superoxide dismutase
Pseudoaltero- monas haloplanktis
3 Site-directed mutagenesis
Tm increased from 48 to 52 °C and an enhanced stability
against GdnHCl
Loop
Increased electrostatic interactions Halostabilizing
NAD- dependent DNA ligase
Finegoldia
magna 1 Site-directed mutagenesis
Improves the
halophilic adaptation Helix
Salt-dependent stabilizing effect in the mesophilic
proteins Acidostabilizing
Carboxyl- esterase
Sulfolobus
tokodaii 3 Site-directed mutagenesis
At pH 3 and 4, the Tm values is reduced due to disruption of the H-
bond or ionic interaction
Loop
Disruption of Hydrogen bond
or ionic interaction Alkalistabilizing
Glycosidase Trichoderma
reesei 1
PCR overlap extension
method
pH optimum is more
alkaline in the mutant Loop
Histidine residue are establishing
the correct electrostatic
micro- environment Barostabilizing
IgG binding- Protein G
Streptococcus
sp. 2 Site-directed
mutagenesis
Hydrogen bonds shorten by 0.022 ± 0.108 Å over
2 kbar (i.e.,
∼0.01 Å/kbar)
Loop
High pressure causes distortion in the secondary
structure
1.9 Stabilizing proteins by non-protein engineering methods
Other than the intrinsic properties of a protein there exist other extrinsic methods for attaining extreme-stability. The engineering of glycosylation is one of the most common posttranslational modifications that stabilize proteins. Its effect on the thermodynamics and kinetics of proteins is poorly understood. The protein stability increases as the degree of glycosylation increases and, to a much smaller extent, with the size of the polysaccharides205.The stabilization effect depends on the position of the glycans; thus,
the same degree of glycosylation may result in a different thermal effect depending on the location of the sugars. Importantly, thermodynamic stabilization is accompanied by kinetic stabilization, with the unfolding barrier increased by ≈20% for the highly glycosylated protein variants. It results in a higher enthalpy206. Olsen and Thomsen observed the effect of glycosylation on thermostability of two Bacillus β-glucanases that were expressed in E. coli and in S. cerevisiae. The one that expressed in S. cerevisiaewas strongly kinetically stabilized by glycosylation at 70 °C, and its optimum temperature for activity was higher207. These examples suggest that glycosylation could represent an alternative method for protein thermostabilization. Similarly, methylation of lysine is also protein stabilization step in posttranslational modification. Edmondson and co-workers reported the monomethylated Lys 5 and Lys7 of native small DNA binding protein Sac7d Sulfolobus acidocaldarius cause stabilization up to 100 °C208.
Inorganic salts are also stabilizing proteins in two ways. First is through a specific effect, where a metal ion interacts with the protein in a conformational manner, and second is through a general salt effect, which mainly affects the water activity. Correspondingly, the general effects of inorganic salt include chaotropic and kosmotropic effects. The 'kosmotrope' (order-maker) solutes stabilize proteins by reducing the solubility of hydrophobic aggregates whereas 'chaotrope' (disorder-maker) solutes destabilize the proteins by disrupting hydrophobic aggregates and increase their solubility leading to unfolding of proteins209. Protein-stabilizing solutes (kosmotropes) increase the extent of hydrogen bonding. Example includes trimethylamine N-oxide, proline, ectoine, α,α- trehalose, glycine betaine and 3-dimethylsulfoniopropionate. Ectoine and its derivative hydroxyectoine are well-known kosmotropic solutes that are widely employed by many of the extremophiles as protein stabilizers210,211 and osmostress protectants212,213. Interestingly, it is reported in some of the extremophile that the ectoine and hydroxyectoine biosynthesis is enhanced by up-regulation of ectoine synthesizing enzymes by extremes in growth temperature, pH and salts environment214–217. Interestingly, Thauer and coworkers reported that five Methanopyrus kandleri methanogenic enzymes have become thermostable and their activity increases by higher concentrations of inorganic salts218.
The physical factor like pressure is also involved in the stabilization of proteins. Like, hyperthermophiles survive under high-temperature environments and also survive under high-pressure environments. Because of these environments, hyperthermophiles cannot avoid pressure and temperature (such as Thermococcus barophilus). All their macromolecular cell components have to be adapted to high pressures219. In such microorganisms, the enzymes are stabilized and activated by high pressures (e.g., M.
jannaschii protease and hydrogenase). The reason behind their stabilization by pressure says that the pressure favors the structure with the smallest volume and high percentage of hydrophobic interactions219. Since pressure played an important role in stabilization of enzymes, it has great potential benefits for activation in biocatalysis. Michels and Clark found that increase in pressure to 500 atm resulted in 3.4-fold and 2.7-fold increase in activity and thermostability, of M. jannaschii protease and hydrogenase, respectively219. In addition, enzyme activity and thermostability increased with pressure: raising the pressure to 500 atm increased the reaction rate at 125 °C 3.4-fold and the thermostability 2.7-fold. Spin labeling of the active-site serine revealed that the active-site geometry of the M. jannaschii protease is not grossly different from that of several mesostable proteases; however, the active-site structure may be relatively rigid at moderate temperatures. The barostable and thermostable behavior of the enzyme is consistent with the barophilic growth of M. jannaschii observed previously219.
1.10 Vivid role of chaperones and extremolytes in stabilizing proteins in extreme