Protein stability and molecular adaptation to extreme conditions.

Proteins, due to the delicate balance of stabilizing and destabilizing interactions, are only marginally stable. Adaptation to extreme environments tends to shift the 'mesophilic' characteristics of proteins to the respective extremes of temperature, hydrostatic pressure, pH and salinity, such that, under the mutual physiological conditions, the molecular properties are similar regarding overall topology, flexibility and solvation. Enhanced intrinsic stability requires only minute local structural changes so that general strategies of stabilization cannot be established. Apart from mutative changes of amino-acid sequences, extrinsic factors (or cellular components) may be involved in 'extremophilic adaptation'. The molecular basis of acidophilic, alkalophilic and barophilic adaptation is still obscure. Mechanisms of enhanced thermal stability involve improved packing density, as well as specific local interactions. In halophiles, water and salt binding of the intrinsically stable protein inventory is accomplished by favoring acidic over basic amino acid residues and decreased hydrophobicity. General limits of viability are: (a) the susceptibility of the covalent structure of the polypeptide chain toward hydrolysis or hydrothermal degradation; (b) the competition of extreme solvent parameters with the weak electrostatic and hydrophobic interactions involved in protein stabilization; (c) perturbations of the folding and assembly of proteins; and (d) 'dislocation' of biochemical pathways due to effects of extreme conditions on the intricate network of metabolic reactions.     

Barophiles: deep-sea microorganisms adapted to an extreme environment

The deep-sea environment is characterized by high pressure and low temperature but in the vicinity of hydrothermal vents regions of extremely high temperature exist. Deep-sea microorganisms have specially adapted features that enable them to live and grow in this extreme environment. Recent research on the physiology and molecular biology of deep-sea barophilic bacteria has identified pressure-regulated operons and shown that microbial growth is influenced by the relationship between temperature and pressure in the deep-sea environment.     

Molecular adaptation and the origin of land plants

The origin and diversification of land plants was one of the most important biological radiations. Land plants are crucial components of all modern terrestrial ecosystems. The first land plants had to adapt to a wide array of new environmental challenges including desiccation, varying temperatures, and increased UV radiation. There have been numerous studies of the morphological adaptations to life on land. However the molecular adaptations to life on land have only recently gained attention. These studies have greatly benefited from the recent advances in our understanding of the phylogenetic relationships between and among the charophycean algae and the basal land plant groups. In this review I summarize the current knowledge of a variety of physiological and biochemical adaptations to land including plant growth hormones, isoprene, phenolics, and heat shock proteins.     

Sequence specific thermal stability of the collagen triple helix

Theoretical calculations of the thermal stability of collagen triple helices using empirical values for the contribution of individual tripeptide units are presented and compared with direct measurements of the thermal stability of various types of collagens. Relative stabilities are assigned to the positions of the tripeptide units in the amino acid sequence along the length of the collagen molecule. The sequence specific relative stabilities of type I and type XI collagens are compared. These offer insight into the reasons for the existence of unfolding intermediates in type XI collagen that are absent in type I collagen. The pattern of relative stabilities calculated for mouse type IV collagen is consistent with experimental results which indicate that the amino terminal region is very stable and that the interruptions cause increased flexibility and independently unfolding domains. Mutations in the triple helical domain of human type I procollagen occurring in brittle bone disease (osteogenesis imperfecta) show varying effects on the thermal stability of the molecule. The sequence specific thermal stability calculations shed some light on why some mutations of cysteine for glycine have greater effects on the thermal stability than others.     

Surface salt bridges contribute to the extreme thermal stability of an FN3-like domain from a thermophilic bacterium

This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (?Tm ?19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.     

Activity,stability and flexibility in glycosidases adapted to extreme thermal environments

To elucidate the strategy of low temperature adaptation for a cold-adapted family 8 xylanase, the thermal and chemical stabilities, thermal inactivation, thermodependence of activity and conformational flexibility, as well as the thermodynamic basis of these processes, were compared with those of a thermophilic homolog. Differential scanning calorimetry, fluorescence monitoring of guanidine hydrochloride unfolding and fluorescence quenching were used, among other techniques, to show that the cold-adapted enzyme is characterized by a high activity at low temperatures, a poor stability and a high flexibility. In contrast, the thermophilic enzyme is shown to have a reduced low temperature activity, high stability and a reduced flexibility. These findings agree with the hypothesis that cold-adapted enzymes overcome the quandary imposed by low temperature environments via a global or local increase in the flexibility of their molecular edifice, with this in turn leading to a reduced stability. Analysis of the guanidine hydrochloride unfolding, as well as the thermodynamic parameters of irreversible thermal unfolding and thermal inactivation shows that the driving force for this denaturation and inactivation is a large entropy change while a low enthalpy change is implicated in the low temperature activity. A reduced number of salt-bridges are believed to be responsible for both these effects. Guanidine hydrochloride unfolding studies also indicate that both family 8 enzymes unfold via an intermediate prone to aggregation.     

Hydroxyproline content and location in relation to collagen thermal stability.

A new analysis has been made on studies of the influence of imino acid content on the changes of collagen thermal stability (t_m). It is shown that, for the interstitial vertebrate collagens, there is a strict regularity in the changes of t_m depending on hydroxyproline content. No correlation is observed between t_m and proline content. Also, no correlation between t_m and hydroxyproline content is observed for invertebrate and basement membrane collagens. On the basis of the reported data, the dependence of t_m on hydroxyproline content is considered to be not a correlation between t_m and the total content of hydroxyproline, but only as the correlation between t_m and the content of hydroxyproline occurring at the third position in the sequence (Gly-R₂-R₃)_n. The results agree with the idea that the influence exerted by proline and hydroxyproline on the stabilization of the triple helix of collagen is different.     

Molecular mechanisms underlying thermal adaptation of xeric animals

For many years, we and our collaborators have investigated the adaptive role of heat shock proteins in different animals, including the representatives of homothermic and poikilothermic species that inhabit regions with contrasting thermal conditions. Adaptive evolution of the response to hyperthermia has led to different results depending upon the species. The thermal threshold of induction of heat shock proteins in desert thermophylic species is, as a rule, higher than in the species from less extreme climates. In addition, thermoresistant poikilothermic species often exhibit a certain level of heat shock proteins in cells even at a physiologically normal temperature. Furthermore, there is often a positive correlation between the characteristic temperature of the ecological niche of a given species and the amount of Hsp70-like proteins in the cells at normal temperature. Although in most cases adaptation to hyperthermia occurs without changes in the number of heat shock genes, these genes can be amplified in some xeric species. It was shown that mobile genetic elements may play an important role in the evolution and fine-tuning of the heat shock response system, and can be used for direct introduction of mutations in the promoter regions of these genes.     

Keeping your options open: Maintenance of thermal plasticity during adaptation to a stable environment

Phenotypic plasticity may allow species to cope with environmental variation. The study of thermal plasticity and its evolution helps understanding how populations respond to variation in temperature. In the context of climate change, it is essential to realize the impact of historical differences in the ability of populations to exhibit a plastic response to thermal variation and how it evolves during colonization of new environments. We have analyzed the real‐time evolution of thermal reaction norms of adult and juvenile traits in Drosophila subobscura populations from three locations of Europe in the laboratory. These populations were kept at a constant temperature of 18ºC, and were periodically assayed at three experimental temperatures (13ºC, 18ºC, and 23ºC). We found initial differentiation between populations in thermal plasticity as well as evolutionary convergence in the shape of reaction norms for some adult traits, but not for any of the juvenile traits. Contrary to theoretical expectations, an overall better performance of high latitude populations across temperatures in early generations was observed. Our study shows that the evolution of thermal plasticity is trait specific, and that a new stable environment did not limit the ability of populations to cope with environmental challenges.     

Age-related thermal stability and susceptibility to proteolysis of rat bone collagen.

The shrinkage temperature (Ts) and the pepsin-solubilizability of collagen fibrils in bone matrix obtained from decalcified femur diaphysis from 2-, 5-, 15- and 25-month-old rats were found to decrease with age. Digestion with human fibroblast collagenase dissolved less than half of the collagen, whereas sequential treatment by pepsin followed by collagenase resulted in its complete dissolution. This result shows that collagenase and a telopeptide-cleaving enzyme, when acting in an appropriate sequence, have a great potential for the degradation of bone collagen. The 'melting' profile of the pepsin-solubilized collagen showed a biphasic transition with transition peak at 35.9 degrees C and 40.8 degrees C. With increasing age an increasing proportion of the collagen 'melted' in the transition peak at 35.9 degrees C (pre-transition), and the 'melting' temperature (Tm) of the collagen decreased in parallel with Ts in relation to age. Both Ts and Tm decreased by 3 degrees C in the age span investigated. The age-related change in Ts could therefore be accounted for by the decrease in molecular stability. The collagenase-cleavage products of the bone collagen obtained by the sequential treatment with pepsin and collagenase showed only one peak transition (at 35.1 degrees C), and the Tm for the products was independent of age. The results indicate that the pre-transition for the pepsin-solubilized collagen is due to an age-related decrease in thermal stability may have implications for the mechanical strength and turnover of the bone collagen. In contrast with bone collagen, soft-tissue collagen showed neither the age-dependency of thermal stability nor the characteristic biphasic 'melting' profile.     

Molecular stability of chemically modified collagen triple helices

Ionic residues influence the stability of collagen triple helices and play a relevant role in the spontaneous aggregation of fibrillar collagens. Collagen types I and II and some of their CNBr peptides were chemically modified in mild conditions with two different protocols. Primary amino groups of Lys and Hyl were N-methylated by formaldehyde in reducing conditions or N-acetylated by sulfosuccinimidyl acetate. The positive charge of amino groups at physiological pH was maintained after the former modification, whereas it was lost after the latter. These chemical derivatizations did not significantly alter the stability of the triple helical conformation of peptide trimeric species. Also the enthalpic change on denaturation was largely unaffected by derivatizations. This implies that no significant variation of weak bonds, either in number or overall strength, and of entropy occur on modification. These properties can probably be explained by the fact that chemically modified groups maintain the ability to form hydrogen bonds.     

Protein thermal stability: the role of protein structure and aqueous environment

A comprehensive bioinformatic analysis was performed on all protein homologous pairs from mesophilic and thermophilic microorganisms present in the RCSB Protein Data Bank in order to yield a clue on the role of protein structure and aqueous environment. Subsequently self-assembly and LB studies were carried out at increasing temperature by nanogravimetry with thermostable thioredoxin (Trx) from Alicyclobacillus acidocaldarius (BacTrx) versus the mesophilic Escherichia coli counterpart (EcTrx). The comparison with earlier 3D atomic structure determined on the same proteins by X-ray crystallographic diffraction and nuclear magnetic resonance confirm the role inner bound water in determining protein thermostability, as suggested by the bioinformatic and nanogravimetric analysis. The above comparative characterizations in protein solution, thin film and crystal allow to draw a possible coherent explanation for the origin and the molecular mechanisms of both heat stability and radiation resistance in proteins.     

Activity, stability and structural studies of lactate dehydrogenases adapted to extreme thermal environments

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate with concomitant oxidation of NADH during the last step in anaerobic glycolysis. In the present study, we present a comparative biochemical and structural analysis of various LDHs adapted to function over a large temperature range. The enzymes were from Champsocephalus gunnari (an Antarctic fish), Deinococcus radiodurans (a mesophilic bacterium) and Thermus thermophilus (a hyperthermophilic bacterium). The thermodynamic activation parameters of these LDHs indicated that temperature adaptation from hot to cold conditions was due to a decrease in the activation enthalpy and an increase in activation entropy. The crystal structures of these LDHs have been solved. Pairwise comparisons at the structural level, between hyperthermophilic versus mesophilic LDHs and mesophilic versus psychrophilic LDHs, have revealed that temperature adaptation is due to a few amino acid substitutions that are localized in critical regions of the enzyme. These substitutions, each having accumulating effects, play a role in either the conformational stability or the local flexibility or in both. Going from hot- to cold-adapted LDHs, the various substitutions have decreased the number of ion pairs, reduced the size of ionic networks, created unfavorable interactions involving charged residues and induced strong local disorder. The analysis of the LDHs adapted to extreme temperatures shed light on how evolutionary processes shift the subtle balance between overall stability and flexibility of an enzyme.     

Matrotrophy in the cave molly: an unexpected provisioning strategy in an extreme environment

Maternal provisioning of animal embryos may be entirely through yolk deposited in the unfertilized egg (lecithotrophy) or may include post-fertilization nutrient transfer (matrotrophy) in varying degrees. Current theory suggests that the extent of post-fertilization provisioning is resource-dependent, with higher levels of matrotrophy being advantageous in more productive environments. In this study, we investigated post-fertilization embryo provisioning in a livebearing fish, Poecilia mexicana, from two different habitats (a toxic cave and a non-toxic surface habitat) that impose different energetic demands and therefore differ in resources available for reproduction. We predicted that fish in the benign habitat would be more matrotrophic than those from the toxic cave. We used two different techniques for this assay: (1) the matrotrophy-index analysis (MI) for field-collected fish and (2) both MI and radio-tracer assay for laboratory-reared females. According to the interpretation of the matrotrophy index, both populations are purely lecithotrophic, while the radio-tracer assay found females from both populations to actively transfer nutrients to developing embryos at approximately the same rate. Our results suggest that P. mexicana, which was traditionally classified as lecithotrophic, is capable of incipient matrotrophy, and that matrotrophy can contribute to embryo provisioning even in populations from resource-limited environments. Furthermore, the analysis of laboratory-reared animals provides evidence for a genetic component to the large offspring size in cave mollies, which had so far only been described from the field. Specifically, our results suggest matrotrophy occurs in species interpreted as lecithotrophic using the MI approach. Hence, to avoid misclassification, both techniques should ideally be employed in concert, rather than individually. Finally, our results provide further insights into the possible evolutionary pathway from lecithotrophic oviparity to matrotrophic viviparity.     

Structure in an extreme environment: NMR at high salt

Proteins from halophiles have adapted to challenging environmental conditions and require salt for their structure and function. How halophilic proteins adapted to a hypersaline environment is still an intriguing question. It is important to mimic the physiological conditions of the archae extreme halophiles when characterizing their enzymes, including structural characterization. The NMR derived structure of Haloferax volcanii dihydrofolate reductase in 3.5 M NaCl is presented, and represents the first high salt structure calculated using NMR data. Structure calculations show that this protein has a solution structure which is similar to the previously determined crystal structure with a difference at the N terminus of beta3 and the type of beta-turn connection beta7 and beta8.     

Silicified virus‐like nanoparticles in an extreme thermal environment: implications for the preservation of viruses in the geological record

Biofilms that grow around Gumingquan hot spring (T = 71 °C, pH = 9.2) in the Rehai geothermal area, Tengchong, China, are formed of various cyanobacteria, Firmicutes, Aquificae, Thermodesulfobacteria, Desulfurococcales, and Thermoproteales. Silicified virus‐like nanoparticles, 40–200 nm in diameter, are common inside the microbial cells and the extracellular polymeric substances around the cells. These nanoparticles, which are formed of a core encased by a silica cortex, are morphologically akin to known viruses and directly comparable to silicified virus‐like particles that were produced in biofilms cultured in the laboratory. The information obtained from examination of the natural and laboratory‐produced samples suggests that viruses can be preserved by silicification, especially while they are still encased in their host cells. These results expand our views of virus–host mineral interaction in extreme thermal environments and imply that viruses can be potentially preserved and identified in the geological record.     

Preface to special issue: "Molecular mechanism of the adaptation of terrestrial plants to gravity environment on Earth"]

Organisms borne in the primitive sea about 30 million years ago had evolved in water without a large influence of gravity on earth. About 4 million years ago, the first terrestrial organisms, plants appeared on the land from the sea. The terrestrial plants have adapted to and evolved on the land environment so that they can extend their roots downward in soil and their shoots upward against 1 g gravity. At least two functions that were acquired during the process of evolution helped the terrestrial plants to adapt to gravity environment on earth. One is gravitropism. The other is the reinforcement of the cell wall, particularly the secondary cell wall. In the present feature articles, the molecular mechanism of the adaptation of terrestrial plants to gravity environment on earth will be reviewed, paying special attention to the mechanism of the genetic control of the signaling of gravity stimulus in gravitropism, automorphogenesis, genes involved in auxin transport, gravity effect on cell wall properties and gravimorphogenesis in terrestrial plants.     

Discrete reduction of type I collagen thermal stability upon oxidation

The oxidation of acid-soluble calf skin collagen type I caused by metal-dependent free radical generating systems, Fe(II)/H2O2 and Cu(II)/H2O2, was found to bring down in a specific, discrete way the collagen thermal stability, as determined by microcalorimetry and scanning densitometry. Initial oxidation results in splitting of the collagen denaturational transition into two components. Along with the endotherm at 41 degrees C typical for non-oxidized collagen, a second, similarly cooperative endotherm appears at 35 degrees C and increases in enthalpy with the oxidant concentration and exposure time, while the first peak correspondingly decreases. The two transitions at 35 and 41 degrees C were registered by densitometry as stepwise increases of the collagen-specific volume. Further oxidation results in massive collagen destruction manifested as abolishment of both denaturational transitions. The two oxidative systems used produce identical effects on the collagen stability but at higher concentrations of Cu(II) in comparison to Fe(II). The discrete reduction of the protein thermal stability is accompanied by a decrease of the free amino groups, suggestive of an oxidation attack of the side chains of lysine residues. Since the denaturation temperature of collagen shifts from above to below body temperature (41 degrees C-35 degrees C) upon oxidation, it appears important to account for this effect in a context of the possible physiological implications of collagen oxidation.