Mechanisms for stabilisation and the maintenance of solubility in proteins from thermophiles

Background  

The database of protein structures contains representatives from organisms with a range of growth temperatures. Various properties have been studied in a search for the molecular basis of protein adaptation to higher growth temperature. Charged groups have emerged as key distinguishing factors for proteins from thermophiles and mesophiles.     

Cold shock and adaptation

Adaptation to environmental stresses, such as temperature fluctuation, is essential for the survival of all living organisms. Cellular responses in both prokaryotes and eukaryotes to high temperature include the synthesis of a set of highly conserved proteins known as the heat shock proteins. In contrast to the heat shock response, adaptation to low temperatures has not been as extensively studied. However, a family of cold-inducible proteins is evident in prokaryotes. In addition, most organisms have developed adaptive mechanisms that alter both membrane fluidity and the protein translation machinery at low temperature. This review addresses the different adaptive mechanisms used by a variety of organisms with a focus on the molecular mechanisms of cold adaptation that have recently been identified during the cold shock response in Escherichia coli. BioEssays 20:49–57, 1998. © 1998 John Wiley & Sons, Inc.     

Investigation on the causes of codon and amino acid usages variation between thermophilic Aquifex aeolicus and mesophilic Bacillus subtilis

Base composition, codon usages and amino acid usages have been analyzed by taking 529 orthologous sequences of Aquifex aeolicus and Bacillus subtilis, having different optimal growth temperatures. These two bacteria do not have significant difference in overall GC composition, but GC(1+2) and GC3 levels were found to vary significantly. Significant increments in purine content and GC3 composition have been observed in the coding sequences of Aquifex aeolicus than its Bacillus subtilis counterparts. Correspondence analyses on codon and amino acid usages reveal that variation in base composition actually influences their codon and amino acid usages. Two selection pressures acting on the nucleotide level (GC3 and purine enrichment), causes variation in the amino acid usage differently in different protein secondary structures. Our results suggest that adaptation of amino acid usages in coil structure of Aquifex aeolicus proteins is under the control of both purine increment and GC3 composition, whereas the adaptation of the amino acids in the helical region of thermophilic bacteria is strongly influenced by the purine content. Evolutionary perspectives concerning the temperature adaptation of DNA and protein molecules of these two bacteria have been discussed on the basis of these results.     

Bacillus species proteins involved in spore formation and degradation: from identification in the genome,to sequence analysis,and determination of function and structure

The members of Bacillus species are Gram-positive, ubiquitous spore-forming bacilli. Several genomic sequences have been made available during recent years, including Bacillus subtilis, a model organism among this genus, Bacillus anthracis, and their analyses provided a wealth of information about spore-forming bacteria. Some members of this species can cause serious diseases in livestock and humans. An important pathogen in this group of organisms is B. anthracis, which is the causative agent of anthrax. A summary of the B. subtilis genome information, based on the publicly released sequence, that allowed for the identification and characterization of new and novel proteins of this organism as well as similar proteins from other members of Bacillus species is provided. The primary goal for this work is to present a review of the genome sequence-identified genes that encode proteins involved in the sporulation, germination, and outgrowth processes. These three processes are essential for spore development and later its transformation into a vegetative cell. Additionally, for a few selected examples of the protein products of the identified genes, the application of bioinformatics and modeling tools is illustrated in order to determine their likely structure and function. Two three-dimensional models of the structures of such proteins, PrfA endonuclease and phosphatase PhoE, are presented together with the structure-based functional conclusions. The review of such studies provides an example of how the genomic sequence can be utilized in order to elucidate the structure and function of proteins, in particular proteins of the Bacillus species. Because only a limited number of proteins of Bacillus species organisms are involved in the synthesis and degradation of spores and have been characterized to date, this genome-based analysis may provide new insights into the developmental processes of bacterial organism.     

Maximal stabilities of reversible two-state proteins

The hydrophobic effect is the major force driving protein folding. Around room temperature, small organic solutes and hydrophobic amino acids have low solubilities in water and the hydrophobic effect is the strongest. These facts suggest that globular proteins should be maximally stable around room temperature. While this fundamental paradigm has been expected, it has not actually been shown to hold. Toward this goal, we have collected and analyzed experimental thermodynamic data for 31 proteins that show reversible two-state folding <--> unfolding transitions at or near neutral pH. Twenty-six of these are unique, and 20 of the 26 are maximally stable around room temperature irrespective of their structural properties, the melting temperature, or the living temperatures of their source organisms. Their average temperature of maximal stability is 293 +/- 8 K (20 +/- 8 degrees C). These proteins differ in size, fold, and number of domains, hydrophobic folding units, and oligomeric states. They derive from the cold-loving psychrophiles, from mesophiles, and from thermophiles. Analysis of the single-domain proteins present in this set shows that the variations in their thermodynamic parameters are correlated in a way which may explain the adaptation of the proteins to the living temperatures of the organisms from which they derive. The average energetic contribution of the individual amino acids toward protein stability decreases with an increase in protein size, suggesting that there may be an upper limit for protein maximal thermodynamic stability. For the remaining proteins, deviation of the maximal stability temperatures from room temperature may be due to greater uncertainties in their heat capacity change (DeltaC(p)) values, a weaker hydrophobic effect, and/or a stronger electrostatic contribution.     

Phylogenetic divergence of fish and mammalian metallothionein: relationships with structural diversification and organismal temperature

Metallothioneins (MTs) are nonenzymatic low molecular weight proteins, that play an important role in the homeostasis and detoxification of heavy metals in a large variety of organisms. These proteins are endowed with striking features, including an unusual amino acid composition characterized by the presence of 20 cysteines out of a total of 60 residues and absence of secondary structure elements. It is generally accepted that MTs underwent few modifications during evolution because of these structural and functional constraints. Such a conclusion is founded on the studies carried out mostly on MTs of mammalian origin. For such a reason, we have decided to compare the MTs of homeothermic and poikilothermic organisms, such as mammals and fish, with the specific aim to put in relation phylogenetic divergence and structural/functional adaptation to temperature. We have included in our analysis also Antarctic Notothenioids, a fish group characterized by genetic isolation and cold-adaptation to a particular harsh environment. We have determined the average hydropathic index of ancestral MT sequences and used them to infer the temperatures of the environment housing the hypothetical ancestor organisms. Finally, we have derived phylogenetic relationships of MT molecules from the pairwise comparison of their three-dimensional structures.     

Physiological responses in a variable environment: relationships between metabolism, hsp and thermotolerance in an intertidal-subtidal species

Physiological responses to temperature reflect the evolutionary adaptations of organisms to their thermal environment and the capability of animals to tolerate thermal stress. Contrary to conventional metabolism theory, increasing environmental temperatures have been shown to reduce metabolic rate in rocky-eulittoral-fringe species inhabiting highly variable environments, possibly as a strategy for energy conservation. To study the physiological adaptations of an intertidal-subtidal species to the extreme and unpredictable heat stress of the intertidal zone, oxygen consumption rate and heat shock protein expression were quantified in the sea cucumber Apostichopus japonicus. Using simulate natural temperatures, the relationship between temperature, physiological performance (oxygen consumption and heat shock proteins) and thermotolerance were assessed. Depression of oxygen consumption rate and upregulation of heat shock protein genes (hsps) occurred in sequence when ambient temperature was increased from 24 to 30°C. Large-scale mortality of the sea cucumber occurred when temperatures rose beyond 30°C, suggesting that the upregulation of heat shock proteins and mortality are closely related to the depression of aerobic metabolism, a phenomenon that is in line with the concept of oxygen- and capacity-limited thermal tolerance (OCLTT). The physiologically-related thermotolerance of this sea cucumber should be an adaptation to its local environment.     

Temperature‐dependent mutational robustness can explain faster molecular evolution at warm temperatures,affecting speciation rate and global patterns of species diversity

Distribution of species across the Earth shows strong latitudinal and altitudinal gradients with the number of species decreasing with declining temperatures. While these patterns have been recognized for well over a century, the mechanisms generating and maintaining them have remained elusive. Here, we propose a mechanistic explanation for temperature‐dependent rates of molecular evolution that can influence speciation rates and global biodiversity gradients. Our hypothesis is based on the effects of temperature and temperature‐adaptation on stability of proteins and other catalytic biomolecules. First, due to the nature of physical forces between biomolecules and water, stability of biomolecules is maximal around + 20°C and decreases as temperature either decreases or increases. Second, organisms that have adapted to cold temperatures have evolved especially flexible (but unstable) proteins to facilitate catalytic reactions in cold, where molecular movements slow down. Both these effects should result in mutations being on average more detrimental at cold temperatures (i.e. lower mutational robustness in cold). At high temperatures, destabilizing water–biomolecule interactions, and the need to maintain structures that withstand heat denaturation, should decrease mutational robustness similarly. Decreased mutational robustness at extreme temperatures will slow down molecular evolution, as a larger fraction of new mutations will be removed by selection. Lower mutational robustness may also select for reduced mutation rates, further slowing down the rate of molecular evolution. As speciation requires the evolution of epistatic incompatibilities that prevent gene flow among incipient species, slow rate of molecular evolution at extreme temperatures will directly slow down the rate at which new species arise. The proposed mechanism can thus explain why molecular evolution is faster at warm temperatures, contributing to higher speciation rate and elevated species richness in environments characterized by stable and warm temperatures.     

Thermodynamic stability and folding of proteins from hyperthermophilic organisms

Life grows almost everywhere on earth, including in extreme environments and under harsh conditions. Organisms adapted to high temperatures are called thermophiles (growth temperature 45-75 degrees C) and hyperthermophiles (growth temperature >or= 80 degrees C). Proteins from such organisms usually show extreme thermal stability, despite having folded structures very similar to their mesostable counterparts. Here, we summarize the current data on thermodynamic and kinetic folding/unfolding behaviors of proteins from hyperthermophilic microorganisms. In contrast to thermostable proteins, rather few (i.e. less than 20) hyperthermostable proteins have been thoroughly characterized in terms of their in vitro folding processes and their thermodynamic stability profiles. Examples that will be discussed include co-chaperonin proteins, iron-sulfur-cluster proteins, and DNA-binding proteins from hyperthermophilic bacteria (i.e. Aquifex and Theromotoga) and archea (e.g. Pyrococcus, Thermococcus, Methanothermus and Sulfolobus). Despite the small set of studied systems, it is clear that super-slow protein unfolding is a dominant strategy to allow these proteins to function at extreme temperatures.     

Biomineralization proteins: from vertebrates to bacteria

Biomineralization processes are frequently found in nature. Living organisms use various strategies to create highly ordered and hierarchical mineral structures under physiologic conditions in which the temperatures and pressures are much lower than those required to form the same mineralized structures by chemical synthesis. Although the mechanism of biomineralization remains elusive, proteins have been found responsible for the formation of such mineral structures in many cases. These proteins are active components in the process of biomineralization. The mechanisms by which their function can vary from providing active organic matrices that control the formation of specific mineral structures to being catalysts that facilitate the crystallization of certain metal ions. This review summarizes the current understanding of the functions of several representative biomineralization proteins from vertebrates to bacteria in the hopes of providing useful insight and guidance for further elucidation of mechanisms of biomineralization processes in living organisms.     

Patterns of temperature adaptation in proteins from Methanococcus and Bacillus

It has long been known that amino acid substitutions in proteins of organisms living at moderate and high temperatures (mesophiles and thermophiles, respectively) are not all symmetrical; for example, more aligned sites have lysine in mesophiles and arginine in thermophiles than have the opposite pattern. This is generally taken to indicate that certain amino acids are favored over others by selection at different temperatures. Previous comparisons of protein sequences from mesophiles and thermophiles have used relatively small numbers of sequences from a diverse array of species, meaning that only the most common amino acid substitutions could be examined and any taxon-specific patterns would be obscured. Here, we compare a large number of proteins between mesophiles and thermophiles in the archaeal genus Methanococcus and the bacterial genus Bacillus. Each genus exhibits dramatically asymmetrical substitution patterns for many pairs of amino acids. There are several pairs of amino acids for which one amino acid is favored in thermophilic Bacillus and the other is favored in thermophilic Methanococcus; this appears to result from the higher G + C content of the DNA of thermophilic Bacillus, a complication not seen in Methanococcus.     

Patterns of temperature adaptation in proteins from the bacteria Deinococcus radiodurans and Thermus thermophilus

Asymmetrical patterns of amino acid substitution in proteins of organisms living at moderate and high temperatures (mesophiles and thermophiles, respectively) are generally taken to indicate selection favoring different amino acids at different temperatures due to their biochemical properties. If that were the case, comparisons of different pairs of mesophilic and thermophilic taxa would exhibit similar patterns of substitutional asymmetry. A previous comparison of mesophilic versus thermophilic Methanococcus with mesophilic versus thermophilic Bacillus revealed several pairs of amino acids for which one amino acid was favored in thermophilic Bacillus and the other was favored in thermophilic Methanococcus. Most of this could be explained by the higher G+C content of the DNA of thermophilic Bacillus, a phenomenon not seen in the Methanococcus comparison. Here, I compared the mesophilic bacterium Deinococcus radiodurans and its thermophilic relative Thermus thermophilus, which are similar in G+C content. Of the 190 pairs of amino acids, 83 exhibited significant substitutional asymmetry, consistent with the pervasive effects of selection. Most of these significantly asymmetrical pairs of amino acids were asymmetrical in the direction predicted from the Methanococcus data, consistent with thermal adaptation resulting from universal biochemical properties of the amino acids. However, 12 pairs of amino acids exhibited asymmetry significantly different from and in the opposite direction of that found in the Methanococcus comparison, and 21 pairs of amino acids exhibited asymmetry that was significantly different from that found in the Bacillus comparison and could not be explained by the greater G+C content in thermophilic Bacillus. This suggests that selection due to universal biochemical properties of the amino acids and differences in G+C content are not the only causes of substitutional asymmetry between mesophiles and thermophiles. Instead, selection on taxon-specific properties of amino acids, such as their metabolic cost, may play a role in causing asymmetrical patterns of substitution.     

Comparative genome analysis of Bacillus cereus group genomes with Bacillus subtilis

Genome features of the Bacillus cereus group genomes (representative strains of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis sub spp. israelensis) were analyzed and compared with the Bacillus subtilis genome. A core set of 1381 protein families among the four Bacillus genomes, with an additional set of 933 families common to the B. cereus group, was identified. Differences in signal transduction pathways, membrane transporters, cell surface structures, cell wall, and S-layer proteins suggesting differences in their phenotype were identified. The B. cereus group has signal transduction systems including a tyrosine kinase related to two-component system histidine kinases from B. subtilis. A model for regulation of the stress responsive sigma factor sigmaB in the B. cereus group different from the well studied regulation in B. subtilis has been proposed. Despite a high degree of chromosomal synteny among these genomes, significant differences in cell wall and spore coat proteins that contribute to the survival and adaptation in specific hosts has been identified.     

Variation of specific proteins, mitochondria and fatty acid composition in gill of Scylla serrata (Crustacea, Decapoda) under low temperature adaptation

The mud crab Scylla serrata is an important commercial crustacean inhabiting estuarine water along the coast of southeast China. Metabolism in the gill is affected continuously by fluctuating water temperature and, therefore, the ability to cope with temperature change is essential to maintain physiological function. This experiment was conducted to help understand the mechanism of low temperature adaptation in S. serrata gill. In this study, 40 healthy juvenile male S. serrata from the same broodstock were grouped randomly into four groups, which were kept at 5 °C, 10 °C, 15 °C and 27 °C, with the same feeding regime during a 3-week adaptation period. Two-dimensional electrophoresis of the proteome was conducted to separate the specific proteins responsible for low temperature adaptation. Variations in the mitochondria were observed using transmission electron microscopy, and fatty acid composition was determined using gas chromatography. The results showed that different numbers of specific proteins were expressed under different low temperature adaptation, with more expressed at 5 °C and 10 °C than at 15 °C. Mitochondrial morphology also varied under different low temperature adaptation, but there was no linear relationship between microbial density and adaptation temperature. The composition of different fatty acids in the gill varied considerably with adaptation temperature, but elongation of the carbon chain and transition from fatty acids occurred at lower temperatures. Thus, changes in the specific proteins, mitochondria and fatty acid composition of the gill were the positive effects of low temperature on metabolism, leading to improved adaptation ability in S. serrata.     

Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey

BACKGROUND: Proteins from thermophilic organisms usually show high intrinsic thermal stability but have structures that are very similar to their mesophilic homologues. From prevous studies it is difficult to draw general conclusions about the structural features underlying the increased thermal stability of thermophilic proteins. RESULTS: In order to reveal the general evolutionary strategy for changing the heat stability of proteins, a non-redundant data set was compiled comprising all high-quality structures of thermophilic proteins and their mesophilic homologues from the Protein Data Bank. The selection (quality) criteria were met by 64 mesophilic and 29 thermophilic protein subunits, representing 25 protein families. From the atomic coordinates, 13 structural parameters were calculated, compared and evaluated using statistical methods. This study is distinguished from earlier ones by the strict quality control of the structures used and the size of the data set. CONCLUSIONS: Different protein families adapt to higher temperatures by different sets of structural devices. Regarding the structural parameters, the only generally observed rule is an increase in the number of ion pairs with increasing growth temperature. Other parameters show just a trend, whereas the number of hydrogen bonds and the polarity of buried surfaces exhibit no clear-cut tendency to change with growth temperature. Proteins from extreme thermophiles are stabilized in different ways to moderately thermophilic ones. The preferences of these two groups are different with regards to the number of ion pairs, the number of cavities, the polarity of exposed surface and the secondary structural composition.     

Purification,crystal structure determination and functional characterization of type III antifreeze proteins from the European eelpout Zoarces viviparus

Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform, are here characterized and their crystal structures determined. We conclude that the higher activity of the QAE1 isoforms cannot be attributed to single residues, but rather a combination of structural effects. Furthermore both ZvAFP6 and ZvAFP13 crystal structures have water molecules around T18 equivalent to the tetrahedral-like waters previously identified in a neutron crystal structure. Interestingly, ZvAFP6 forms dimers in the crystal, with a significant dimer interface. The presence of ZvAFP6 dimers was confirmed in solution by native electrophoresis and gel filtration. To our knowledge this is the first report of dimerization of AFP type III proteins.     

Spin-labeling studies on the lipids of psychrophilic, psychrotrophic, and mesophilic clostridia.

Spin-labeling studies were conducted to elucidate the viscosity and phase transition temperatures of lipids isolated from psychrophilic, psychrotrophic, and mesophilic clostridia. Electron spin resonance spectroscopy indicated that the lipids, for all the growth temperatures tested, were in a fluid state and from 13 to 24 C higher than the corresponding lipid transition temperatures. When the organisms were grown at different temperatures, a psychrotropic and two mesophilic clostridia were shown to be able to adjust their lipid-phase transition temperature to the growth temperature. A psychrophilic Clostridium strain, when grown at different temperatures, synthesized lipids that had the same phase transition temperature. It is suggested that this lack of growth temperature-inducible regulation of lipid-phase transition temperature may be a molecular determinant for the psychrophily of this organism. It is proposed that the growth temperature range of an organism is dependent upon the ability of the organism to regulate its lipid fluidity within a specific range.     

Bacillus Species Proteins Involved in Spore Formation and Degradation: From Identification in the Genome,to Sequence Analysis,and Determination of Function and Structure

The members of Bacillus species are Gram-positive, ubiquitous spore-forming bacilli. Several genomic sequences have been made available during recent years, including Bacillus subtilis, a model organism among this genus, Bacillus anthracis, and their analyses provided a wealth of information about spore-forming bacteria. Some members of this species can cause serious diseases in livestock and humans. An important pathogen in this group of organisms is B. anthracis, which is the causative agent of anthrax. A summary of the B. subtilis genome information, based on the publicly released sequence, that allowed for the identification and characterization of new and novel proteins of this organism as well as similar proteins from other members of Bacillus species is provided. The primary goal for this work is to present a review of the genome sequence-identified genes that encode proteins involved in the sporulation, germination, and outgrowth processes. These three processes are essential for spore development and later its transformation into a vegetative cell. Additionally, for a few selected examples of the protein products of the identified genes, the application of bioinformatics and modeling tools is illustrated in order to determine their likely structure and function. Two three-dimensional models of the structures of such proteins, PrfA endonuclease and phosphatase PhoE, are presented together with the structure-based functional conclusions. The review of such studies provides an example of how the genomic sequence can be utilized in order to elucidate the structure and function of proteins, in particular proteins of the Bacillus species. Because only a limited number of proteins of Bacillus species organisms are involved in the synthesis and degradation of spores and have been characterized to date, this genome-based analysis may provide new insights into the developmental processes of bacterial organism.