Thermophiles are the microorganism that are adaptive to high temperatures. They are normally found only in hot springs, hydrothermal vents and similar sites of geothermal activity. Their intrinsically thermostable enzymes offers advantages for industrial and diagnostic processes ranging from ore processing to molecular genotyping. These microorganisms include diverse archaea and bacteria, and they span a wide range of metabolic strategies (Grogan et al. 1998). Various molecular features enable the cells of extreme thermophiles to function optimally at temperatures that kill other cells.Thermophilic adaptations can be observed to be the most versatile among all being capable to utilize majority of the stabilization features such as increases fluidity of cell membranes, produces chaperones, increases and decreases the expression of selected genes, elaborating novel genes and proteins that address thermal stress-specific concerns such as chaperonins (Hsp70, Hsp90 and Hsp100 chaperone system), alters the kinetic and regulatory properties of enzymes and functional proteins, etc. (Heimburg, 2007). Thus, different factors are responsible to stabilize all macromolecules of thermophiles in different extreme conditions. These factors can be studied in four hierarchical levels: i.e. genome sequence, amino acid sequence and protein structure (Chakravorty et al. 2017).
Factors responsible for Thermostability
A large repertoire of thermophilic organisms has been identified. However, only few of them have been successfully sequenced. Search for genomes with the key words "thermophilic" and "hyperthermophilic" in NCBI genome search results in a total of 156 hits for thermophiles and hyperthermophiles. Based on these genome search results it can be demonstrated that most of the work to understand extreme-stability has been carried out for thermophiles. It has been reported that variations in nucleotide composition can have very significant effects on the patterns of codon usage and thermostability (Frank and Lobry 1999). Thermophiles and hyperthermophiles have been reported to have a high GC content (Bao et al. 2002; Saunders et al. 2003). The comparison of base composition, codon and amino acid usages of thermophilic Aquifex aeolicus with those of Bacillus subtilis shows that there is a significant increase in purine content and GC composition in codon selection. This type of alteration in base compositions also influences the codon and amino acid usages of A. aeolicus (Basak et al. 2004). Further it was reported that codon usage preferences might be based on an error-minimizing selection at the protein level. Mutation occurs in any codon for thermophilic adaptation, which results in similar amino acids that leads to the similar protein conformations which are relatively less deleterious compared to others. For example, Arg codon changing to Lys codon has less deleterious effect in protein conformations because of similar properties of both amino acids (Najafabadi et al. 2005). Arginine is coded by two sets of codons AGR (AGA and AGG) and CGN (CGU, CGC, CGA and CGG). The biasness is also seen in between these two sets of arginine codons AGR and CGN. Whenever there is an increase in lysine for thermophilic adaptations, AGR codons are more preferred codons for arginine coding (Farias and Bonato 2003). Because a positive error minimization mechanism between arginine and lysine codons (AAA and AAG) occurs due to a single mutation at second position that could turn AGR arginine codons into codons for lysine (which can be harmful for thermostability). Whereas the decrease in CGN codon usage in organisms that live in high temperatures can be interpreted as a negative error minimization (Farias and Bonato 2003). Since a single mutation at second position could turn CGN arginine codons into codons for histidine (CAU and CAC) and glutamine (CAA and CAG) which can be harmful for thermostability (Farias and Bonato 2003). Work carried out by Zeldovich et al. (2007) concludes that an increase in purine (A+G) of thermophilic bacterial genomes due to the preference for isoleucine, valine, tyrosine, tryptophan, arginine, glutamine, and leucine, which have purine-rich codon patterns, is responsible for the possible primary adaptation mechanism for thermophilicity (Zeldovich et al. 2007). These amino acid residues increase the content of hydrophobic and charged amino acids, enhancing thermostability. Additionally, DNA was observed to possess positive supercoils resulting in greater thermostability (Madigan, 2000). These studies help in understanding the use of preferred codon in an thermophilic organism as powerful tools that can be utilized to improve function predictions and genome-environment mappings (Goodarzi et al. 2008). But, there is still lack of knowledge of thermophile genomes and global codon usage. Further, codon usage studies were done for thermophiles but with no conclusive finding as which codon is preferred in thermophiles.
Many researchers have attempted to study the effect of amino acids on protein thermostability (Karshikoff and Ladenstein 1998; Spassov et al. 1995; Szilagyi and Zavodszky 2000; Vogt et al. 1997). At temperatures greater than 100oC the thermostability of amino acids in proteins have been reported by Jaenicke and Bohm (1998) as follows (V, L) > I> Y> K> H> M> T> S> W> (D, E, R, C). An interesting rule was derived by De Farias and Bonato (2003) who found that the E+K/Q+H ratio can distinguish hyperthermophiles (>4.5) from mesophiles (< 2.5) and thermophiles (3.2-4.6). Trivedi et al. (2006) said that "as there are variations in preference for other amino acids between mesophiles, thermophiles and hyperthermophiles, it is apparent that these variations are not only organism specific but are also protein specific within the organism" (Trivedi et al. 2006).
Protein destabilization at higher temperatures has been reported mainly due to the presence of thermolabile residues as they are prone to deamidation, cleavage of peptide bonds and oxidation of Cys and Met residues. Deamidation of Asn residues is a spontaneous and non-enzymatic reaction which can occur in acidic, neutral, or alkaline conditions. Therefore proteins with high Asn residues are suspected to be less thermostable (Kumar et al. 2000). Furthermore reports also suggest that Asn containing peptides are more prone to deamidation at a faster rate than glutamine counterparts (Parthiban 2006). The cleavage of a peptide can destabilize proteins by disrupting a protein chain. Shirley 1995 identified three modes of peptide bond cleavage. This can occur by (1) hydrolysis of peptide bonds under acidic conditions at Asp residues, (2) succinimide formation at Asn residues at physiological pH, and (3) proteolysis by enzymes. Literature survey shows that protein having high frequency of Asp and Asn residues were less thermostable (Kumar et al. 2000).
Oxidation of Cys and Met often results in protein destabilization. Cys has been reported to be involved in protein stability of extracellular proteins by formation of disulphide bridges. Such covalent interactions have been found in many thermostable proteins. Formation of disulphide bond is dependent on the protein conformation. However free Cys residues are prone to oxidation and β-elimination (Whitaker et al. 1983). This has been shown to irreversibly inactivate ribonuclease and lysozyme at pH 6-8 and 90-100° (Ahern and Klibanov 1988). Similarly, Met oxidation has been associated with the inactivation of proteins (Swaim and Pizzo 1988). Many thermostable proteins have been reported to have low frequency of Met residues (Kumar et al. 2000; Mattos 2002).
Enhancement of intra-protein or Van der Waals interactions has been reported to be associated with increasing thermostability of proteins (Reetz 2013; Trivedi et al. 2006). Hydrogen bonds are the most abundant type of noncovalent interactions, and 4-12 kJ mol/1 is required to break a single bond (Vieille and Zeikus 2001). The hydrogen bonding is the highest cited feature in literature for protein thermostabilization (Vogt et al. 1997). In thermostable proteins hydrogen bonds shows an increase of 11.7 hydrogen bonds per chain per 10° rise in thermostability (Vogt et al. 1997). It was reported that surface charged and polar side chains with high conformational mobility can form alternative hydrogen bonded donor-acceptor pairs (Khechinashvili et al. 2006). The conclusion drawn was that residues located in the N- and C-terminal regions and in the extended loops that are capable of forming alternative longer range H-bonded pairs, leads to enhance the protein thermostability (Khechinashvili et al. 2006). Hydrogen bonds can be further divided into the following types depending on their donor and acceptor atoms as main chain-main chain, main chain-side chain and side chain-side chain hydrogen bonds. The other types of hydrogen bonds that have been classified are charge-charge and charge-neutral hydrogen bonds. Charge-neutral hydrogen bonds are more stabilizing as desolvation energy making for an H-bond residue is lower than for an ion pair (Tanner et al. 1996). Moreover binding energy of a charged-neutral H-bond is far larger than from neutral-neutral H-bonds, due to the charge-dipole interaction. A study of 16 protein families shows that thermostable proteins show a consistent increase in hydrogen bonds (Vogt et al. 1997). They can also be divided into: short strong hydrogen bonds: Distance 0.20-0.25nm. They acquire covalent characteristics and are also known as low barrier hydrogen bonds. N-H-O, O-H-O, N-H-N hydrogen bonds are said to be higher in energy than other types of hydrogen bonds and biologically more important (Panigrahi et al. 2015). Recently Srivastava et al. in 2014 showed that increase in hydrogen bond increases thermostability of Bacillus subtilis lipases as predicted through molecular dynamics simulations and network-based analysis (Srivastava and Sinha 2014).
Electrostatic interactions have long been implicated in the thermostability of thermophilic proteins (Szilagyi and Zavodszky 2000; Vogt and Argos 1997; Vogt et al. 1997). Theoretical studies suggest that the stabilizing effect of electrostatic interactions increases with increasing temperature (Panja et al. 2015; Matsuura et al. 2015). It was reported that thermostable proteins tend to have more salt bridges and surface charge residues (Fukuchi et al. 2003). Salt bridges are formed by spatially proximal pairs of oppositely charged residues in native protein structures. A salt bridge is constituted by a couple of oppositely charged groups, so in proteins it is recognized if at least one Asp or Glu side-chain carboxyl oxygen atom (i.e. OD in Asp or OE in Glu) and one side-chain nitrogen atom of Arg, Lys or His (i.e. NH in Arg, NZ in Lys or NE & ND in His) are within a distance of 4.0 Angstroms (Costantini et al. 2008). A single salt bridge can contribute 13-22 kJ/mol to the free energy of folding (Jaenicke 1996) and, unlike hydrophobic interactions (Privalov and Gill 1988), they are relatively unaffected at extremely high temperatures. Elcock proposed that salt-bridge should be more stabilizing at high temperatures because the unfavorable desolvation penalty (Elcock 1998) and the entropic cost of fixing two charged side-chains would decrease with temperatures. Furthermore, thermostability is achieved by upshifting or broadening the thermostability curve which is obtained by Differential Scanning Calorimeter (DSC). A smaller ΔCp can increase the maximum ΔGu where, ΔGu(Ts) = ΔHm-ΔCp (Tm-Ts), or in other words, the protein stability curve is up-shifted if ΔHm is increased or remains constant (Elcock 1998; Kumar et al. 2000). Salt bridges decrease ΔCp thus results in the upshift of the thermostability curve (Chan et al. 2011). The effect of ionic interactions on thermostability has been studied by loss of function and gain of function mutations. Vetriani et al. 1998 reported that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes (Vetriani et al. 1998). They conclude this by studying structures of hexameric glutamate dehydrogenases (GluDHs) from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis. Schmid and co-workers have implicated contributions of electrostatic interactions to the thermostability of thermophilic Bacillus caldolyticus cold shock protein.
Cation-π interaction is another form of electrostatic interaction responsible for protein thermostability. They have been reported to be formed by the interactions between positively charged residues (Lys and Arg) and aromatic amino acids (Tyr, Trp and Phe). Gromiha et al. (2002) analyzed the influence of cation-π interactions to enhance the stability from mesostable to thermostable proteins. Tyr has a greater number of such interactions with Lys in thermostable proteins. The influence of Phe in making cation-π interactions is higher in mesophiles than in thermophiles (Gromiha et al. 2002). Further, a network of cation-π interactions is maintained by Lys in thermostable proteins, whereas Arg plays a major role in mesostable proteins. Moreover, atoms that have a substantial positive charge in both Lys and Arg make a more significant contribution for cation-π interactions than do cationic group atoms (Gromiha et al. 2004). The cation-π interaction between Arg19 and Tyr93 in the protein indole-3-glycerol phosphate synthase from Sulfolobus solfataricus was reported to contribute towards stability (Knochel et al. 1996).
Hydrophobic interactions have been reported by many authors to play crucial role in protein folding. It is brought about by burial of solvated non-polar side chains. Each additional methyl group buried in the enzyme gives an increase in stability of 1.3 (±0.5) kcal mol/1 (Pace, 1992). An enhanced hydrophobic effect is one of the reported reasons for the slow unfolding of thermostable proteins (Okada et al. 2010). Rathi et al. (2015) studied a set of 130 pairs of thermostable and mesostable proteins and reported hydrophobic interactions as "key factors" for protein thermostability (Rathi et al. 2015). Burg et al. (1994) increased thermostability of thermolysin-like neutral protease of Bacillus stearothermophilus by introducing Arg, Lys or bulky hydrophobic amino acids (BURG et al. 1994). Through their experiments it was shown that surface hydrophobic contacts were the major determinants for protein thermostability. Unfortunately mutations attempting to fill cavities often were found to be not that stabilizing due to detrimental effects of unfavorable Van der Waals interactions and subsequent local rearrangements (Vieille and Zeikus 2001). Furthermore it has been reported that hydrophobic interactions, which are entropic at room temperature but becomes enthalpic at higher temperature, reaches their maximum stabilizing effect at 75° (Makhatadze and Privalov 1995). Core packing is often linked to increased hydrophobicity and stability (Schumann et al. 1993). An increase in hydrophobicity, given that it being buried will add to core stability due to increased Van der Waals interactions. Programs such as ROC, PROSE, PERLA and CORE can be used to redesign protein cores using molecular force fields. Many proteins were successfully redesigned using these methods. However sometimes over packing of the cores results in destabilization of the folded conformer (Ventura et al. 2002). Core packing also results in rigidity of proteins. Rigidity has also been related to enhance thermostability of proteins. Rigidity causes hyperthermostable enzymes to be often inactive at low temperatures (Vieille and Zeikus 2001).
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