Thermodynamic activation properties of elongation factor 2 (EF-2) proteins from psychrotolerant and thermophilic Archaea

In this study, the thermodynamic activation parameters of cold-adapted proteins from Archaeaa are described for the first time for the irreversible protein unfolding and ribosome-dependent GTPase activity of elongation factor 2 (EF-2) from the psychrotolerant Methanococcoides burtonii and the thermophilic Methanosarcina thermophila. Thermolability of Methanococcoides burtonii EF-2 was demonstrated by a low activation free-energy of unfolding as a result of low activation-enthalpy. Although structural data for EF-2 are presently limited to protein homology modeling, the observed thermodynamic properties are consistent with a low number of noncovvalent bonds or an altered solvent interaction, causing a loss of entropy during the unfolding process. A physiological concentration of potassium aspartate or potassium glutamate was shown to stabilize both proteins against irreversible denaturation by strengthening noncovalent interactions, as indicated by increased activation enthalpies. The transition state of GTPase activity for Methanococcoides burtonii EF-2 was characterized by a lower activation enthalpy than for Methanosarcina thermophila EF-2. The relative entropy changes could be explained by differential displacement of water molecules during catalysis, resulting in similar activation free energies for both proteins. The presence of solutes was shown to facilitate the breaking of enthalpy-driven interactions and structuring of more water molecules during the reaction. By studying the thermodynamic activation parameters of both GTPase activity and unfolding and examining the effects of intracellular solutes and partner proteins (ribosomes), we were able to identify enthalpic and entropic properties that have evolved in the archaeal EF-2 proteins to enable Methanococcoides burtonii and Methanosarcina thermophila to adapt to their respective thermal environments.     

Effect of temperature on stability and activity of elongation factor 2 proteins from Antarctic and thermophilic methanogens

Despite the presence and abundance of archaea in low-temperature environments, little information is available regarding their physiological and biochemical properties. In order to investigate the adaptation of archaeal proteins to low temperatures, we purified and characterized the elongation factor 2 (EF-2) protein from the Antarctic methanogen Methanococcoides burtonii, which was expressed in Escherichia coli, and compared it to the recombinant EF-2 protein from a phylogenetically related thermophile, Methanosarcina thermophila. Using differential scanning calorimetry to assess protein stability and enzyme assays for the intrinsic GTPase activity, we identified biochemical and biophysical properties that are characteristic of the cold-adapted protein. This includes a higher activity at low temperatures caused by a decrease of the activation energy necessary for GTP hydrolysis and a decreased activation energy for the irreversible denaturation of the protein, which indicates a less thermostable structure. Comparison of the in vitro properties of the proteins with the temperature-dependent characteristics of growth of the organisms indicates that additional cytoplasmic factors are likely to be important for the complete thermal adaptation of the proteins in vivo. This is the first study to address thermal adaptation of proteins from a free-living, cold-adapted archaeon, and our results indicate that the ability of the Antarctic methanogen to adapt to the cold is likely to involve protein structural changes.     

Unsaturated diether phospholipids in the Antartic methanogen Methanococcoides burtonii

Abstract The Antarctic methanogen Methanococcoides burtonii contained only diether phospholipids. These membrane components were analysed by gas chromatography and gas chromatography mass spectrometry. Of particular interest was the occurrence of unsaturated diether lipids in M. burtonii ; unsaturated ether lipids accounted for 57% of the diether phospholipids. To our knowledge, unsaturated ether lipids have not been previously reported in a methanogen. The presence of the unsaturated ether lipids in M. burtonii is probably the result of temperature adaptation by the bacterium. It may be possible to use these components as a chemical signature for methanogens in Antarctic and Southern Ocean environments.     

Defining the response of a microorganism to temperatures that span its complete growth temperature range (-2°C to 28°C) using multiplex quantitative proteomics

The growth of all microorganisms is limited to a specific temperature range. However, it has not previously been determined to what extent global protein profiles change in response to temperatures that incrementally span the complete growth temperature range of a microorganism. As a result it has remained unclear to what extent cellular processes (inferred from protein abundance profiles) are affected by growth temperature and which, in particular, constrain growth at upper and lower temperature limits. To evaluate this, 8-plex iTRAQ proteomics was performed on the Antarctic microorganism, Methanococcoides burtonii. Methanococcoides burtonii was chosen due to its importance as a model psychrophilic (cold-adapted) member of the Archaea, and the fact that proteomic methods, including subcellular fractionation procedures, have been well developed. Differential abundance patterns were obtained for cells grown at seven different growth temperatures (-2°C, 1°C, 4°C, 10°C, 16°C, 23°C, 28°C) and a principal component analysis (PCA) was performed to identify trends in protein abundances. The multiplex analysis enabled three largely distinct physiological states to be described: cold stress (-2°C), cold adaptation (1°C, 4°C, 10°C and 16°C), and heat stress (23°C and 28°C). A particular feature of the thermal extremes was the synthesis of heat- and cold-specific stress proteins, reflecting the important, yet distinct ways in which temperature-induced stress manifests in the cell. This is the first quantitative proteomic investigation to simultaneously assess the response of a microorganism to numerous growth temperatures, including the upper and lower growth temperatures limits, and has revealed a new level of understanding about cellular adaptive responses.     

Characterization of the ribosomal properties required for formation of a GTPase active complex with the eukaryotic elongation factor 2

The binding stability of the different nucleotide-dependent and -independent interactions between elongation factor 2 (EF-2) and 80S ribosomes, as well as 60S subunits, was studied and correlated to the kinetics of the EF-2- and ribosome-dependent hydrolysis of GTP. Empty reconstituted 80S ribosomes were found to contain two subpopulations of ribosomes, with approximately 80% capable of binding EF-2.GuoPP[CH2]P with high affinity (Kd less than 10(-9) M) and the rest only capable of binding the factor-nucleotide complex with low affinity (Kd = 3.7 x 10(-7) M). The activity of the EF-2- and 80S-ribosome dependent GTPase did not respond linearly to increasing factor concentrations. At low EF-2/ribosome ratios the number of GTP molecules hydrolyzed/factor molecule was considerably lower than at higher ratios. The low response coincided with the formation of the high-affinity complex. At increasing EF-2/ribosome ratios, the ribosomes capable of forming the high-affinity complex was saturated with EF-2, thus allowing formation of the low-affinity ribosome.EF-2 complex. Simultaneously, the GTPase activity/factor molecule increased, indicating that the low-affinity complex was responsible for activating the GTP hydrolysis. The large ribosomal subunits constituted a homogeneous population that interacted with EF-2 in a low-affinity (Kd = 1.3 x 10(-6) M) GTPase active complex, suggesting that the ribosomal domain responsible for activating the GTPase was located on the 60S subunit. Ricin treatment converted the 80S particles to the type of conformation only capable of interacting with EF-2 in a low-affinity complex. The structural alteration was accompanied by a dramatic increase in the EF-2-dependent GTPase activity. Surprisingly, ricin had no effect on the factor-catalyzed GTP hydrolysis in the presence of 60S subunits alone.     

Chaperonins from an Antarctic archaeon are predominantly monomeric: crystal structure of an open state monomer

Archaea are abundant in permanently cold environments. The Antarctic methanogen, Methanococcoides burtonii, has proven an excellent model for studying molecular mechanisms of cold adaptation. Methanococcoides burtonii contains three group II chaperonins that diverged prior to its closest orthologues from mesophilic Methanosarcina spp. The relative abundance of the three chaperonins shows little dependence on organism growth temperature, except at the highest temperatures, where the most thermally stable chaperonin increases in abundance. In vitro and in vivo, the M. burtonii chaperonins are predominantly monomeric, with only 23-33% oligomeric, thereby differing from other archaea where an oligomeric ring form is dominant. The crystal structure of an N-terminally truncated chaperonin reveals a monomeric protein with a fully open nucleotide binding site. When compared with closed state group II chaperonin structures, a large-scale ≈ 30° rotation between the equatorial and intermediate domains is observed resulting in an open nucleotide binding site. This is analogous to the transition observed between open and closed states of group I chaperonins but contrasts with recent archaeal group II chaperonin open state ring structures. The predominance of monomeric form and the ability to adopt a fully open nucleotide site appear to be unique features of the M. burtonii group II chaperonins.     

Protein adaptations to temperature and pressure: complementary roles of adaptive changes in amino acid sequence and internal milieu

Retention of required structural and functional properties of proteins in species adapted to different temperatures and pressures is achieved through variation in amino acid sequence and accumulation of small organic solutes that stabilize protein traits. Conservation of ligand binding and catalytic rate can be achieved by minor differences in sequence. For orthologs of lactate dehydrogenase-A (A₄-LDH) temperature adaptation may involve only a single amino acid substitution. Adaptation involves changes in conformational mobility of regions of A₄-LDH that undergo movement during ligand binding, movements that are rate-limiting to catalysis. A model that integrates adaptations in sequence and intracellular milieu is developed on the basis of conformational microstates. Although orthologs of different thermally adapted species vary in stability, at physiological temperatures it is hypothesized that a similar ensemble of conformational microstates exists for all orthologs. Organic solutes stabilize this ensemble of microstates. Differences among orthologs in responses to organic solutes at a common temperature lead to similar responses at normal body temperatures. Because protein stability increases at high protein concentrations, intrinsic stabilities of proteins may reflect the protein concentrations of the cellular compartments in which they occur. Protein–stabilizing solutes like trimethylamine-N-oxide (TMAO) conserve protein function and structure at elevated hydrostatic pressures.     

Some properties and the possible role of intrinsic ATPase of rat liver 80S ribosomes in peptide bond elongation

The properties and role in peptide elongation of ATPase intrinsic to rat liver ribosomes were investigated. (i) Rat liver 80S ribosomes showed high ATPase and GTPase activities, whereas the GTPase activity of EF-1alpha and EF-2 was very low. mRNA, aminoacyl-tRNA, and elongation factors alone enhanced ribosomal ATPase activity and in combination stimulated it additively or synergistically. The results suggest that these translational components induce positive conformational changes of 80S ribosomes by binding to different regions of ribosomes. Translation inhibitors, tetracyclin and fusidic acid, inhibited ribosomal ATPase with or without elongational components. (ii) Two ATPase inhibitors, AMP-P(NH)P and vanadate, did not inhibit GTPase activities of EF-1alpha and EF-2 assayed as uncoupled GTPase, but they did inhibit poly(U)-dependent polyphe synthesis of 80S ribosomes. (iii) Effects of AMP-P(NH)P and ATP on poly(U)-dependent polyphe synthesis at various concentrations of GTP were examined. ATP enhanced the activity of polyphe synthesis even at high concentrations of GTP, suggesting a specific role of ATP. At low concentrations of GTP, the extent of inhibition by AMP-P(NH)P was very low, probably owing to the prevention of the reduction of the GTP concentration. (iv) Vanadate inhibited the translocation reaction by high KCl-washed polysomes. These findings together indicate that ribosomal ATPase participates in peptide translation by inducing positive conformational changes of mammalian ribosomes, in addition to its role of chasing tRNA from the E site.     

Ice-active proteins from the Antarctic nematode Panagrolaimus davidi

The Antarctic nematode Panagrolaimus davidi has an ice-active protein that shows recrystallization inhibition but no thermal hysteresis. It belongs to a class of ice-active proteins found in a variety of freezing-tolerant organisms that display insignificant levels of thermal hysteresis in the context of the environmental temperatures to which they are exposed. The recrystallization inhibition activity of the P. davidi ice-active protein is present at low concentrations, is relatively heat stable, is affected more by acid than by alkaline pH, is not calcium dependant and is not affected by reagents that target carbohydrate residues or sulphydryl linkages. A hexagonal ice crystal growth form also indicates the presence of an ice-active protein. This protein could have important functions in the survival of intracellular freezing by this organism by controlling the stability of ice after its formation.     

Studies of the GTPase domain of archaebacterial ribosomes

Ribosomes from the methanogens Methanococcus vannielii and Methanobacterium formicicum catalyse uncoupled hydrolysis of GTP in the presence of factor EF-2 from rat liver (but not factor EF-G from Escherichia coli). In this assay, and in poly(U)-dependent protein synthesis, they were sensitive to thiostrepton. In contrast, ribosomes from Sulfolobus solfataricus did not respond to factor EF-2 (or factor EF-G) but possessed endogenous GTPase activity, which was also sensitive to thiostrepton. Ribosomes from the methanogens did not support (p)ppGpp production, but did appear to possess the equivalent of protein L11, which in E. coli is normally required for guanosine polyphosphate synthesis. Protein L11 from E. coli bound well to 23S rRNA from all three archaebacteria (as did thiostrepton) and oligonucleotides protected by the protein were sequenced and compared with rRNA sequences from other sources.     

Biliproteins and phycobilisomes from cyanobacteria and red algae at the extremes of habitat

This review considers the properties of biliproteins from cyanobacteria and red algae that grow in extreme habitats. Three situations are presented: cyanobacteria that grow at high temperatures; a red alga that grows in acidic conditions at high temperature; and an Antarctic red alga that grows in the cold in dim light conditions. In particular, the properties of their biliproteins are compared to those from organisms from more usual environments. C-phycocyanins from two cyanobacteria able to grow at high temperatures are found to differ in their stabilities when compared to C-phycocyanin from mesophilic algae. They differ in opposite ways, however. One is more stable to dissociation than the mesophilic protein, and the other is more easily dissociated at low temperatures. The thermophilic proteins resist thermal denaturation much better than the mesophilic proteins. The most thermophilic cyanobacterium has a C-phycocyanin with a unique blue-shifted absorption maximum which does not appear to be part of the adaptation of the cyanobacterium to high temperature. The C-phycocyanin from the high-temperature red alga is able to resist dissociation better than mesophilic C-phycocyanins. Electron micrographs show the phycobilisomes of these algae. The Antarctic alga grows under ice at some distance down the water column. Its R-phycoerythrin has a novel absorption spectrum that gives the alga an improved ability to harvest blue light. This may enhance its survival in its light-deprived habitat.     

Protein structure and function at low temperatures

Proteins represent the major components in the living cell that provide the whole repertoire of constituents of cellular organization and metabolism. In the process of evolution, adaptation to extreme conditions mainly referred to temperature, pH and low water activity. With respect to life at low temperatures, effects on protein structure, protein stability and protein folding need consideration. The sequences and topologies of proteins from psychrophilic, mesophilic and thermophilic organisms are found to be highly homologous. Commonly, adaptive changes refer to multiple alterations of the amino acid sequence, which presently cannot be correlated with specific changes of structure and stability; so far it has not been possible to attribute specific increments in the free energy of stabilization to well-defined amino-acid exchanges in an unambiguous way. The stability of proteins is limited at high and low temperatures. Their expression and self-organization may be accomplished under conditions strongly deviating from optimum growth conditions. Molecular adaptation to extremes of temperature seems to be accompanied by a flattening of the temperature profile of the free energy of stabilization. In principle, the free energy of stabilization of proteins is small compared to the total molecular energy. As a consequence, molecular adaptation to extremes of physical conditions only requires marginal alterations of the intermolecular interactions and packing density. Careful statistical and structural analyses indicate that altering the number of ion pairs and hydrophobic interactions allows the flexibility of proteins to be adjusted so that full catalytic function is maintained at varying temperatures.     

Studies on the high molecular weight form of polypeptide chain elongation factor-1 from pig liver. III. Temperature-dependent dissociation into subunits in the presence of GTP

Dissociation of highly purified EF-1 alpha beta gamma (a high molecular weight form of polypeptide chain elongation factor-1) from pig liver into EF-1 alpha and EF-1 beta gamma at various temperatures was examined and the following results were obtained. (i) When dissociation of EF-1 alpha beta gamma was analyzed by gel filtration with Sephacryl S-200, it was found that in the absence of GTP, it did not dissociate at any temperature between 4 and 37 degrees C, whereas in the presence of GTP, it tended to dissociate with elevation of the temperature, and almost complete dissociation was observed at 32 degrees C. This indicated that the dissociation constant of EF-1 alpha beta gamma into EF-1 alpha and EF-1 beta gamma in the presence of GTP increased with increase in the temperature. (ii) When gel filtration was performed in the presence of both GTP and [14C]Phe-tRNA, the formation of a ternary complex of EF-1 alpha . GTP . [14C]Phe-tRNA from EF-1 alpha beta gamma was noted, and its amount was found to increase with elevation of the temperature. (iii) The amount of [14C]Phe-tRNA bound to ribosomes dependent on added EF-1 alpha beta gamma similarly increased with increase in the temperature, as in the case of ternary complex formation, whereas the binding of [14C]Phe-tRNA to ribosomes dependent on free EF-1 alpha proceeded fairly well even at 0 degrees C. From these results we concluded that among the reaction steps in the binding of [14C]Phe-tRNA to ribosomes dependent on EF-1 alpha beta gamma, dissociation of EF-1 alpha beta gamma to form EF-1 alpha . GTP and EF-1 beta gamma in the presence of GTP is the step which is strongly influenced by temperature.     

Purification and characterization of three elongation factors, EF-1 alpha, EF-1 beta gamma, and EF-2, from wheat germ

Three elongation factors, EF-1 alpha, EF-1 beta gamma and EF-2, have been isolated from wheat germ. EF-1 alpha and EF-2 are single polypeptides with molecular weights of approximately 52,000 and 102,000, respectively. The most highly purified preparations of EF-1 beta gamma contain four polypeptides with molecular weights of approximately 48,000, 46,000 and 36,000, 34,000. EF-1 alpha supports poly(U)-directed binding of Phe-tRNA to wheat germ ribosomes and catalyzes the hydrolysis of GTP in the presence of ribosomes, poly(U), and Phe-tRNA. EF-2 catalyzes the hydrolysis of GTP in the presence of ribosomes alone and is ADP-ribosylated by diphtheria toxin to the extent of 0.95 mol of ADP-ribose/mol of EF-2. EF-1 beta gamma decreases the amount of EF-1 alpha required for polyphenylalanine synthesis about 20-fold. EF-1 beta gamma enhances the ability to EF-1 alpha to support the binding of Phe-tRNA to the ribosomes and enhances the GTPase activity of EF-1 alpha. Wheat germ EF-1 alpha, EF-1 beta gamma, and EF-2 support polyphenylalanine synthesis on rabbit reticulocyte ribosomes as well as on yeast ribosomes.     

Compatible solute effects on thermostability of glutamine synthetase and aspartate transcarbamoylase from Methanococcus jannaschii

Methanococcus jannaschii accumulates alpha- and beta-glutamate as osmolytes. The effect of these and other solutes on the thermostability of two multisubunit metabolic enzymes from M. jannaschii, aspartate transcarbamoylase catalytic trimer (ATCase C3) and glutamine synthetase (GS), has been measured and compared to solute effects on bacterial mesophilic counterparts in order to explore if osmolytes accumulated by each organism can preferentially stabilize the proteins to thermal unfolding. For both ATCase enzymes and for the B. subtilis GS, the solutes normally accumulated by the organism were very effective in protecting the enzyme from losing activity at high temperatures, although solute effects on loss of secondary structure did not necessarily correlate with this thermoprotection of activity. The recombinant M. jannaschii GS exhibited quite different behavior. The pure enzyme had a thermal unfolding transition with a midpoint temperature (Tm) less than 60 degrees C, well under the growth temperature of the organism (85 degrees C). None of the small molecule solutes tested (including the K+-glutamate isomers accumulated by M. jannaschii) significantly stabilized the protein to incubation at 85 degrees C. Instead, protein-protein interactions, as illustrated by E. coli GroEL or ribosomal protein L2 stabilization of GS, appeared to be the dominant factor in stabilizing this archaeal enzyme at the growth temperature.     

Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes.

Hygromycin B is an unusual aminoglycoside antibiotic active against both prokaryotic and eukaryotic cells. Hygromycin B at 0.38 mM concentration completely halts yeast cell growth in rich media, presumably by preventing protein synthesis by cytoplasmic ribosomes. Polypeptide synthesis in cell-free extracts from rabbit reticulocytes, wheat germ and yeast is strongly blocked by low concentrations of hygromycin B. The antibiotic inhibits peptide chain elongation by yeast polysomes by preventing elongation factor EF-2-dependent translocation, although it does not affect either the formation of the EF-2-GTP-ribosome complex or the EF-2- and ribosome-dependent GTP hydrolysis which takes place uncoupled from translocation. The inhibition of translocation by hygromycin B might result from the stabilization of peptidyl-tRNA bound to the ribosomal acceptor site, since the stability of [3H]Phe-tRNA-EF-1-poly(U)-ribosome and [3H]Phe-tRNA-poly(U)-ribosome complexes is increased in the presence of hygromycin B. The inhibition of polyphenylalanine synthesis by reticulocyte ribosomes and enzymic translocation of peptidyl-tRNA by yeast polysomes can be reversed by increasing concentrations of EF-2 suggesting a relationship between the binding sites of EF-2 and hygromycin B on the ribosome. Neither non-enzymic translocation, that takes place in the presence of high potassium concentrations, nor the peptide bondforming step are affected by hygromycin B.     

Modification of the accessibility of ribosomal proteins after elongation factor 2 binding to rat liver ribosomes and during translocation

Free- and EF-2-bound 80 S ribosomes, within the high-affinity complex with the non-hydrolysable GTP analog: guanylylmethylenediphosphonate (GuoPP(CH2)P), and the low-affinity complex with GDP, were treated with trypsin under conditions that modified neither their protein synthesis ability nor their sedimentation constant nor the bound EF-2 itself. Proteins extracted from trypsin-digested ribosomes were unambiguously identified using three different two-dimensional gel electrophoresis systems and 5 S RNA release was checked by submitting directly free- and EF-2-bound 80 S ribosomes, incubated with trypsin, to two-dimensional gel electrophoresis. Our results indicate that the binding of (EF-2)-GuoPP[CH2]P to 80 S ribosomes modified the behavior of a cluster of five proteins which were trypsin-resistant within free 80 S ribosomes and trypsin-sensitive within the high-affinity complex (proteins: L3, L10, L13a, L26, L27a). As for the binding of (EF-2)-GDP to 80 S ribosomes, it induced an intermediate conformational change of ribosomes, unshielding only protein L13a and L27a. Quantitative release of free intact 5 S RNA which occurred in the first case but not in the second one, should be related to the trypsinolysis of protein(s) L3 and/or L10 and/or L26. Results were discussed in relation to structural and functional data available on the ribosomal proteins we found to be modified by EF-2 binding.     

Intrinsic versus extrinsic stabilization of enzymes: the interaction of solutes and temperature on A4-lactate dehydrogenase orthologs from warm-adapted and cold-adapted marine fishes.

We examined the effects of temperature and stabilizing solutes on A4-lactate dehydrogenase (A4-LDH) from warm- and cold-adapted fishes, to determine how extrinsic stabilizers affect orthologs with different intrinsic stabilities. Conformational changes during substrate binding are rate-limiting for A4-LDH, thus stabilization due to intrinsic or extrinsic factors leads to decreased activity. A4-LDH from a warm-temperate goby (Gillichthys mirabilis), which has lower values for kcat and the Michaelis constant for pyruvate ( K m PYR), was intrinsically more stable than the orthologs of the cold-adapted Antarctic notothenioids Parachaenichthys charcoti and Chionodraco rastrospinosus, as shown by a higher apparent transition ('melting') temperature (Tm(APP)). We used four solutes, glycerol, sucrose, trimethylamine-N-oxide and poly(ethylene glycol) 8000, which stabilize proteins through different modes of preferential exclusion, to study temperature-solute interactions of the three orthologs. Changes in Tm(APP) were similar for all orthologs in each solute tested, but the catalytic rate of G. mirabilis A4-LDH was decreased most by solutes and increased most by temperature. In contrast, the K m PYR values of the Antarctic orthologs were more affected than that of the goby by both solutes and temperature. We conclude that (a) preferential exclusion of solutes functions within the native state of A4-LDH to favor conformational microstates with minimal surface area; (b) the varied effects of the different solutes on the kinetic properties are due to the interaction between this nonspecific stabilization and the differing intrinsic stabilities of the orthologs; (c) the catalytic rates of A4-LDH orthologs are equally affected by stabilizing solutes, if measurements are made at physiologically appropriate temperatures; and (d) global stability and localized flexibility of these A4-LDH orthologs may evolve independently.     

Enzymes from psychrophilic organisms

Abstract: Psychrophilic organisms such as micro-organisms and other ectothermic species living in polar, deep- sea or any constantly low temperature environments, produce enzymes adapted to function at low temperature. These enzymes are characterized by a high catalytic efficiency at low and moderate temperatures but are rather thermolabile. Due to their high specific activity and their rapid inactivation at temperatures as low as 30°C, they offer, along with the producing micro-organisms, a great potential in biotechnology. The molecular basis of the adaptation of cold α-amylase, subtilisin, triose phosphate isomerase from Antarctic bacteria and of trypsin from fish living in North Atlantic and in Antarctic sea waters have been studied. The comparison of the 3D structures obtained either by protein modelling or by X-ray crystallography (North Atlantic trypsin) with those of their mesophilic counterparts indicates that the molecular changes tend to increase the flexibility of the structure by a weakening of the intramolecular interactions and by an increase of the interactions with the solvent. For each enzyme, the most appropriate strategy enabling it to accommodate the substrate at a low energy cost is selected. There is a price to pay in terms of thermosensibility because the selective pressure is essentially oriented towards the harmonization of the specific activity with ambient thermal conditions. However, as demonstrated by site-directed mutagenesis experiments carried out on the Antarctic subtilisin, the possibility remains to stabilize the structure of these enzymes without affecting their high catalytic efficiency.     

The role of cold-shock proteins in low-temperature adaptation of food-related bacteria

There is a considerable interest in the cold adaptation of food-related bacteria, including starter cultures for industrial food fermentations, food spoilage bacteria and food-borne pathogens. Mechanisms that permit low-temperature growth involve cellular modifications for maintaining membrane fluidity, the uptake or synthesis of compatible solutes, the maintenance of the structural integrity of macromolecules and macromolecule assemblies, such as ribosomes and other components that affect gene expression. A specific cold response that is shared by nearly all food-related bacteria is the induction of the synthesis so-called cold-shock proteins (CSPs), which are small (7 kDa) proteins that are involved in mRNA folding, protein synthesis and/or freeze protection. In addition, CSPs are able to bind RNA and it is believed that these proteins act as RNA chaperones, thereby reducing the increased secondary folding of RNA at low temperatures. In this review established and novel aspects concerning the structure, function and control of these CSPs are discussed. A model for bacterial cold adaptation, with a central role for ribosomal functioning, and possible mechanisms for low-temperature sensing are discussed.