Adaptation of model proteins from cold to hot environments involves continuous and small adjustments of average parameters related to amino acid composition

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Abstract

The growth temperature adaptation of six model proteins has been studied in 42 microorganisms belonging to eubacterial and archaeal kingdoms, covering optimum growth temperatures from 7 to 103 °C. The selected proteins include three elongation factors involved in translation, the enzymes glyceraldehyde-3-phosphate dehydrogenase and superoxide dismutase, the cell division protein FtsZ. The common strategy of protein adaptation from cold to hot environments implies the occurrence of small changes in the amino acid composition, without altering the overall structure of the macromolecule. These continuous adjustments were investigated through parameters related to the amino acid composition of each protein. The average value per residue of mass, volume and accessible surface area allowed an evaluation of the usage of bulky residues, whereas the average hydrophobicity reflected that of hydrophobic residues. The specific proportion of bulky and hydrophobic residues in each protein almost linearly increased with the temperature of the host microorganism. This finding agrees with the structural and functional properties exhibited by proteins in differently adapted sources, thus explaining the great compactness or the high flexibility exhibited by (hyper)thermophilic or psychrophilic proteins, respectively. Indeed, heat-adapted proteins incline toward the usage of heavier-size and more hydrophobic residues with respect to mesophiles, whereas the cold-adapted macromolecules show the opposite behavior with a certain preference for smaller-size and less hydrophobic residues. An investigation on the different increase of bulky residues along with the growth temperature observed in the six model proteins suggests the relevance of the possible different role and/or structure organization played by protein domains. The significance of the linear correlations between growth temperature and parameters related to the amino acid composition improved when the analysis was collectively carried out on all model proteins.

Introduction

Biodiversity in the Earth's biosphere includes a large proportion of organisms called extremophiles, having colonized extreme environments (Nisbet and Sleep, 2001; Cockell and Stokes, 2004). Cold and hot temperatures are hostile habitats for life, and organisms having an optimum growth under the most extreme temperature conditions are named psychrophiles and hyperthermophiles, respectively. Properties of these microorganisms belonging to eubacterial or archaeal kingdom have been extensively reviewed (Stetter, 1996, Stetter, 1998, Stetter, 1999; Hicks and Kelly, 1999; Huber et al., 2000; Deming, 2002; D’Amico et al., 2006; Cavicchioli, 2006). Indeed, it is known that some psychrophiles sustain a residual biological activity even at −20 °C (Deming, 2002), whereas some hyperthermophiles are able to grow up to 113 °C (Blöchl et al., 1997). The increasing discovery and characterization of this type of extremophiles, and the possibility to compare the properties of their biomolecules to that of mesophiles growing at ‘usual’ temperatures, offer the opportunity to study the molecular basis of life adaptation under a wide range of growth temperature.

The main targets in the environmental adaptation of extremophilic sources are proteins, the most abundant flexible macromolecules involved in the control of the whole metabolic pathways and in the structural organization of the microorganism. Several reviews summarized the properties of proteins isolated from thermophiles (Jaenicke and Zavodsky, 1990; Jaenicke, 1991; Adams, 1993; Vieille et al., 1996; Jaenicke and Böhm, 1998; Hicks et al. 1999; Niehaus et al., 1999; Vieille and Zeikus, 2001; Sterner and Liebl, 2001) and psychrophiles (Feller et al., 1997; Feller and Gerday, 1997; Gerday et al. 1997; Saunders et al., 2003; Georlette et al. 2004; Siddiqui and Cavicchioli, 2006). Comprehensive studies on crystal structures of thermophilic proteins did not reveal unusual conformations specific to the source type (Petukhov et al., 1997; Facchiano et al., 1998; Karshikoff and Ladenstein, 1998; Szilagyi and Zavodszky, 2000; Kumar et al., 2000). A similar behavior is observed with psychrophilic proteins (Russell et al., 1998; Maes et al., 1999; Violot et al., 2005), even though the number of crystallographic structures available in this case is much lower. Therefore, it seems that in these extremophilic sources the overall structure of a protein is very similar to that possessed by the mesophilic counterpart, thus reflecting the adaptation of the specific function of the macromolecule, rather than a tolerance to the living environment in the host source. However, hyperthermophilic proteins are endowed with an extraordinary heat stability, as a consequence of a more tight compactness of the protein structure (Vieille and Zeikus, 2001). Vice versa, the psychrophilic counterparts possess an increased protein flexibility, which in most cases leads to a decreased stability compared to mesophilic proteins (D’Amico et al., 2006); in some psychrophilic enzymes the protein flexibility is enhanced in localized regions of the protein structure (Fields and Somero, 1998). Temperature adaptation of proteins is mostly relevant for the catalytic properties of the enzymes, as they must adapt the rate of the catalyzed reaction to the growth temperature of the organism. Indeed, thermophilicity studies revealed that psychrophiles synthesize cold-adapted enzymes endowed with a specific activity at low temperatures, significantly higher compared to that possessed by the mesophilic counterparts (Georlette et al., 2004; Siddiqui and Cavicchioli, 2006). On the other hand, the specific activity of hyperthermophilic enzymes reaches its optimum only at high temperatures, close to the optimum growth conditions of the source (Vieille et al., 1996; Vieille and Zeikus, 2001). Therefore, temperature adaptation of proteins reflects a multifactorial equilibrium between counteracting forces affecting flexibility, stability and activity of proteins. In particular, the similarity of the protein structure and the occurrence of a common catalytic mechanism in proteins isolated from sources adapted from cold to hot environments indicate that the challenge to the extreme environments has been likely accomplished by a fine modulation of the amino acid composition of proteins aimed at optimizing the number of specific weak interactions inside the protein core. Indeed, the amino acid composition has been found to play an important role in determining the protein structural class (Chou and Zhang, 1994, Chou and Zhang, 1995; Chou, 1995; Chou and Maggiora, 1998), in identifying protein subcellular localization, and many other attributes (Chou and Elrod, 1999; Chou, 2002). Evidence has been presented that the amino acid composition in the proteomes of Escherichia coli and Bacillus subtilis reflects a natural selection to enhance metabolic efficiency in these microorganisms (Akashi and Gojobori, 2002). Furthermore, the analysis on several Saccharomyces cerevisiae genes showed a correlation between gene expression level and amino acid composition (Raghava and Han, 2005).

In order to understand the structural requirements for protein adaptation to heat or cold, the amino acid composition of proteins isolated from (hyper)thermophiles (Jaenicke, 1991; Jaenicke and Böhm, 1998; Vieille and Zeikus, 2001; Sterner and Liebl, 2001) or psychrophiles (Feller et al., 1997; Feller and Gerday, 1997; Gerday et al. 1997; Saunders et al., 2003) has long been compared with that of mesophiles. Some of these investigations have been focused on the whole genome of extremophilic microorganisms, thus considering the total protein content of the selected microbial sources. The frequencies of each amino acid residue have been derived from an average amino acid composition, in order to discover a possible bias in the amino acid usage of the considered extremophile. However, each microbial source and each protein seems to adopt only a few of possible, and even counteracting, structural trends (D’Amico et al., 2006; Sterner and Liebl, 2001). For instance, the amino acid bias discovered in some psychrophiles (Saunders et al., 2003) is not applicable to similarly adapted, but evolutionary distant sources (Rabus et al., 2004; Medigue et al., 2005). The ambiguous results are probably related to the different content and/or representation of proteins analyzed for each microbial source. Furthermore, the genetic drift or the natural selection between different microorganisms could hide critical amino acid changes for thermal adaptation (Siddiqui and Cavicchioli, 2006). Some specific key contributions for thermal adaptation of proteins have been proposed through the comparison of the structural and functional properties of proteins in differently adapted sources or through the effect of a mutagenic analysis of a target protein on its thermal stability. These studies led to the discovery of a number of different basic mechanisms involved in thermal stability of thermophilic proteins, as previously reviewed (Vieille and Zeikus, 2001; Sterner and Liebl, 2001). For instance, surface loop depletion, an increased occurrence of hydrophobic residues with branched side chains, and an enhanced proportion of charged residues are apparently the most consistent structural factors contributing to thermostability in thermophilic proteins (Kumar and Nussinov, 2001). Furthermore, on the basis of thermodynamic differences among homologous thermophilic and mesophilic proteins, the higher stability possessed by thermophilic proteins is probably due to specific interactions, particularly electrostatic, present into the protein (Kumar et al., 2001). Finally, the unusual thermal stability of an ATP-binding cassette ATPase of mesophilic origin could be predicted on the basis of its content of polar amino acid residues (Sarin et al., 2003). However, it is not rare that the most important factor for the thermostability of a given protein is not applicable to explain the heat stability of a different one.

This article addresses the question of a possible continuum in the strategy of protein adaptation to the different growth temperatures of the host source. For this reason the amino acid composition of model proteins has been analyzed in several microbial sources displaying an optimum growth temperature ranging from 7 to 103 °C. In particular, we have analyzed the temperature dependence of average parameters related to the amino acid composition. The data obtained suggest that the average values per residue of mass, hydrophobicity, volume and accessible surface, linearly increase with the optimum growth temperature of the microbial source. This finding implies a small variation of the amino acid composition, leading to a moderate bias in the amino acid usage, depending on the growth temperature of the source. Indeed, in (hyper)thermophilic model proteins the content of heavier-size and more hydrophobic residues is increased with respect to mesophilic counterparts; vice versa, smaller-size and less hydrophobic residues are preferred in psychrophilic proteins.

Section snippets

Microbial sources

The 42 microorganisms considered in this study have been chosen for their different adaptation to growth temperature (Table 1). They belong to the living domains of eubacteria (25 sources) and archaea (17 sources), whose complete sequenced genome is available on-line, except for Bacillus stearothermophilus and Pyrococcus woesei. The selected microbial sources, whose respective optimum growth temperatures are indicated in Table 1, include psychrophiles, mesophiles, thermophiles and

Correlation between growth temperature and average parameters related to the amino acid composition in six model proteins from different host microorganisms

Four average parameters related to the amino acid composition of six model proteins were considered to evaluate their dependences on the growth temperature of 42 different microorganisms. In particular, we have chosen the average mass, volume and accessible surface area per residue, because altogether they allow an evaluation of the usage of bulky residues in the amino acid composition. Another parameter considered was the average hydrophobicity, as it reflects the content of hydrophobic

Discussion

Protein adaptation to the growth temperature of host microorganisms should imply the occurrence of small adjustments in the amino acid composition of the macromolecule, that equilibrate the required number of weak interactions, without altering the overall structure of the protein. The bias towards the usage of selected amino acid residues found in some extremophiles reflects the genetic drift of the organism rather than an adaptation to extreme environments. For this reason, the average

Acknowledgments

This work was supported by Grants from the Ministero dell’Università, Istruzione e Ricerca (PRIN 2005, MIUR Italy).

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