Molecular Basis Of Cold Adaptation Research Articles

Identification of hub genes that variate the qCSS12-mediated cold tolerance between indica and japonica rice using WGCNA.

Cold-tolerant QTL qCSS12-regulated 14 hub genes are involved in the chloroplastic biological processes and in the protein synthesis and degradation processes in japonica rice. Low temperature is a main constraint factor for rice growth and production. To better understand the regulatory mechanisms underlying the cold tolerance phenotype in rice, here, we selected a cold-sensitive nearly isogenic line (NIL) NIL(qcss12) as materials to identify hub genes that are mediated by the cold-tolerant locus qCSS12 through weighted gene co-expression network analysis (WGCNA). Fourteen cold-responsive genes were identified, of which, 6 are involved in regulating biological processes in chloroplasts, including the reported EF-Tu, Prk, and ChlD, and 8 are involved in the protein synthesis and degradation processes. Differential expression of these genes between NIL(qcss12) and its controls under cold stress may be responsible for qCSS12-mediated cold tolerance in japonica rice. Moreover, natural variations in 12 of these hub genes are highly correlated with the cold tolerance divergence in two rice subspecies. The results provide deep insights into a better understanding of the molecular basis of cold adaptation in rice and provide a theoretical basis for molecular breeding.

Thirty-degree shift in optimum temperature of a thermophilic lipase by a single-point mutation: effect of serine to threonine mutation on structural flexibility.

In order to understand the molecular basis of cold adaptation, we have used directed evolution to transform a thermophilic lipase LipR1 into its psychrophilic counterpart. A single round of random mutagenesis followed by screening for improved variants yielded a mutant with single-point mutation LipR1M1 (S130T), with optimum activity at 20 °C. Its activity at 50 °C is only 20% as compared to wild type (100%). It showed catalytic rate constant (k cat) 3 times higher and a catalytic efficiency (k cat/K m) 4 times that of wild type. Circular dichroism and fluorescence studies also supported our observation of mutant structural flexibility. Structure analysis using homology models showed that Threonine 130 is exposed to solvent and has lost H-bond interaction with neighboring amino acid, thereby increasing flexibility of this lipase structure.

Psychrophilic Bacteria: Biodiversity, Molecular Basis of Cold Adaptation and Biotechnological Implications

Psychrophilic Bacteria: Biodiversity, Molecular Basis of Cold Adaptation and Biotechnological Implications

Strategy for cold adaptation of the tryptophan synthase α subunit from the psychrophile Shewanella frigidimarina K14-2: crystal structure and physicochemical properties

To investigate the molecular basis of cold adaptation of enzymes, we determined the crystal structure of the tryptophan synthase α subunit (SfTSA) from the psychrophile Shewanella frigidimarina K14-2 by X-ray analysis at 2.6-Å resolution and also examined its physicochemical properties. SfTSA was found to have the following characteristics: (i) The stabilities against heat and denaturant of SfTSA were lower than those of an α subunit (EcTSA) from Escherichia coli. This lower equilibrium stability originated from both a faster unfolding rate and a slower refolding rate; (ii) the heat denaturation of SfTSA was completely reversible at pH 7.0 and the solubility of denatured SfTSA was higher than that of denatured EcTSA. The two-state transition of denaturation for SfTSA was highly cooperative, whereas the denaturation process of EcTSA was considerably more complex and (iii) the global structure of SfTSA was quite similar to those of α subunits from other species. Relative to those other proteins, SfTSA exhibited an increase in cavity volume and a decrease in the number of ion pairs. SfTSA also lacks a hydrogen bond near loop B, related to catalytic function. These characteristics of SfTSA might provide the conformational flexibility required for catalytic activity at low temperatures.

Differential expression of membrane proteins helps Antarctic Pseudomonas syringae to acclimatize upon temperature variations

Antarctic bacteria are adapted to the extremely low temperature. The transcriptional and translational machineries of these bacteria are adapted to the sub-zero degrees of temperature. Studies directed towards identifying the changes in the protein profiles during changes in the growth temperatures of an Antarctic bacterium Pseudomonas syringae Lz4W may help in understanding the molecular basis of cold adaptation. In this study, subcellular fractionation methods of proteins were used for the enrichment and identification of proteins including low abundance proteins. The membrane proteins of the bacterium P. syringae Lz4W were prepared employing sucrose density gradient method. The proteins were separated through 2D gel-electrophoresis with the pH ranges 3-10, 4-7 and 5-8 using the detergent, amidosulfobetaine (ASB-14). The proteins separated on the 1D SDS PAGE and 2D gels were identified with the help of LC-ESI MS/MS and MALDI TOF TOF using bioinformatic programs MASCOT and SEQUEST. Since the genome sequence of P. syringae Lz4W is not available, the proteins are identified by using the genome database of the Pseudomonas sp. available at NCBI. The present studies focus on identifying temperature dependent expression of proteins by employing LC-MS/MS method and the functional significance of these proteins is discussed.

Structure and Dynamics of the First Archaeal Parvulin Reveal a New Functionally Important Loop in Parvulin-type Prolyl Isomerases

Parvulins are a group of peptidyl-prolyl isomerases (PPIases) responsible for important biological processes in all kingdoms of life. The PinA protein from the psychrophilic archaeon Cenarchaeum symbiosum is a parvulin-like PPIase. Due to its striking similarity to the human parvulins Pin1 and Par14, PinA constitutes an interesting subject for structural and functional studies. Here, we present the first high resolution NMR structure of an archaeal parvulin, PinA, based on 1798 conformational restraints. Structure calculation yields an ensemble of 20 convergent low energy structures with a backbone r.m.s.d. value of 0.6 Å within the secondary structure elements. The overall fold of PinA comprises the β-α3-β-α-β2 fold typical for all parvulin structures known so far, but with helix III being a short 310-helix. A detailed comparison of this high resolution structure of the first archaeal PinA protein with bacterial and eukaryotic parvulin PPIase structures reveals an atypically large catalytic binding site. This feature provides an explanation for cold-adapted protein function. Moreover, the residues in and around 310-helix III exhibit strong intramolecular dynamics on a microsecond to millisecond timescale and display structural heterogeneity within the NMR ensemble. A putative peptide ligand was found for PinA by phage display and was used for 1H-15N-HSQC titrations. Again, the flexible region around 310-helix III as well as residues of the peptide binding pocket showed the strongest chemical shift perturbations upon peptide binding. The local flexibility of this region also was modulated by ligand binding. A glycine and two positively charged residues are conserved in most parvulin proteins in this flexible loop region, which may be of general functional importance for parvulin-type PPIases.

Comparative proteome analysis of psychrophilic versus mesophilic bacterial species: Insights into the molecular basis of cold adaptation of proteins

BackgroundCold adapted or psychrophilic organisms grow at low temperatures, where most of other organisms cannot grow. This adaptation requires a vast array of sequence, structural and physiological adjustments. To understand the molecular basis of cold adaptation of proteins, we analyzed proteomes of psychrophilic and mesophilic bacterial species and compared the differences in amino acid composition and substitution patterns to investigate their likely association with growth temperatures.ResultsIn psychrophilic bacteria, serine, aspartic acid, threonine and alanine are overrepresented in the coil regions of secondary structures, whilst glutamic acid and leucine are underrepresented in the helical regions. Compared to mesophiles, psychrophiles comprise a significantly higher proportion of amino acids that contribute to higher protein flexibility in the coil regions of proteins, such as those with tiny/small or neutral side chains. Amino acids with aliphatic, basic, aromatic and hydrophilic side chains are underrepresented in the helical regions of proteins of psychrophiles. The patterns of amino acid substitutions between the orthologous proteins of psychrophiles versus mesophiles are significantly different for several amino acids when compared to their substitutions in orthologous proteins of within the mesophiles or psychrophiles.ConclusionCurrent results provide quantitative substitution preferences (or avoidance) of amino acids that lead to the adaptation of proteins to cold temperatures. These finding would help future efforts in selecting mutations for rational design of proteins with enhanced psychrophilic properties.

Structural adaptation to low temperatures − analysis of the subunit interface of oligomeric psychrophilic enzymes

Enzymes from psychrophiles show higher catalytic efficiency in the 0-20 degrees C temperature range and often lower thermostability in comparison with meso/thermophilic homologs. Physical and chemical characterization of these enzymes is currently underway in order to understand the molecular basis of cold adaptation. Psychrophilic enzymes are often characterized by higher flexibility, which allows for better interaction with substrates, and by a lower activation energy requirement in comparison with meso/thermophilic counterparts. In their tertiary structure, psychrophilic enzymes present fewer stabilizing interactions, longer and more hydrophilic loops, higher glycine content, and lower proline and arginine content. In this study, a comparative analysis of the structural characteristics of the interfaces between oligomeric psychrophilic enzyme subunits was carried out. Crystallographic structures of oligomeric psychrophilic enzymes, and their meso/thermophilic homologs belonging to five different protein families, were retrieved from the Protein Data Bank. The following structural parameters were calculated: overall and core interface area, characterization of polar/apolar contributions to the interface, hydrophobic contact area, quantity of ion pairs and hydrogen bonds between monomers, internal area and total volume of non-solvent-exposed cavities at the interface, and average packing of interface residues. These properties were compared to those of meso/thermophilic enzymes. The results were analyzed using Student's t-test. The most significant differences between psychrophilic and mesophilic proteins were found in the number of ion pairs and hydrogen bonds, and in the apolarity of their subunit interface. Interestingly, the number of ion pairs at the interface shows an opposite adaptation to those occurring at the monomer core and surface.

Flexibility and enzymatic cold-adaptation: A comparative molecular dynamics investigation of the elastase family

Molecular dynamics simulations of representative mesophilic and psycrophilic elastases have been carried out at different temperatures to explore the molecular basis of cold adaptation inside a specific enzymatic family. The molecular dynamics trajectories have been compared and analyzed in terms of secondary structure, molecular flexibility, intramolecular and protein–solvent interactions, unravelling molecular features relevant to rationalize the efficient catalytic activity of psychrophilic elastases at low temperature. The comparative molecular dynamics investigation reveals that modulation of the number of protein–solvent interactions is not the evolutionary strategy followed by the psycrophilic elastase to enhance catalytic activity at low temperature. In addition, flexibility and solvent accessibility of the residues forming the catalytic triad and the specificity pocket are comparable in the cold- and warm-adapted enzymes. Instead, loop regions with different amino acid composition in the two enzymes, and clustered around the active site or the specificity pocket, are characterized by enhanced flexibility in the cold-adapted enzyme. Remarkably, the psycrophilic elastase is characterized by reduced flexibility, when compared to the mesophilic counterpart, in some scattered regions distant from the functional sites, in agreement with hypothesis suggesting that local rigidity in regions far from functional sites can be beneficial for the catalytic activity of psychrophilic enzymes.

Molecular basis of cold adaptation

Cold-adapted, or psychrophilic, organisms are able to thrive at low temperatures in permanently cold environments, which in fact characterize the greatest proportion of our planet. Psychrophiles include both prokaryotic and eukaryotic organisms and thus represent a significant proportion of the living world. These organisms produce cold-evolved enzymes that are partially able to cope with the reduction in chemical reaction rates induced by low temperatures. As a rule, cold-active enzymes display a high catalytic efficiency, associated however, with a low thermal stability. In most cases, the adaptation to cold is achieved through a reduction in the activation energy that possibly originates from an increased flexibility of either a selected area or of the overall protein structure. This enhanced plasticity seems in turn to be induced by the weak thermal stability of psychrophilic enzymes. The adaptation strategies are beginning to be understood thanks to recent advances in the elucidation of the molecular characteristics of cold-adapted enzymes derived from X-ray crystallography, protein engineering and biophysical methods. Psychrophilic organisms and their enzymes have, in recent years, increasingly attracted the attention of the scientific community due to their peculiar properties that render them particularly useful in investigating the possible relationship existing between stability, flexibility and specific activity and as valuable tools for biotechnological purposes.

Cold Adaptation of a Mesophilic Subtilisin-like Protease by Laboratory Evolution

Enzymes isolated from organisms native to cold environments generally exhibit higher catalytic efficiency at low temperatures and greater thermosensitivity than their mesophilic counterparts. In an effort to understand the evolutionary process and the molecular basis of cold adaptation, we have used directed evolution to convert a mesophilic subtilisin-like protease from Bacillus sphaericus, SSII, into its psychrophilic counterpart. A single round of random mutagenesis followed by recombination of improved variants yielded a mutant, P3C9, with a catalytic rate constant (k(cat)) at 10 degrees C 6.6 times and a catalytic efficiency (k(cat)/K(M)) 9.6 times that of wild type. Its half-life at 70 degrees C is 3.3 times less than wild type. Although there is a trend toward decreasing stability during the progression from mesophile to psychrophile, there is not a strict correlation between decreasing stability and increasing low temperature activity. A first generation mutant with a >2-fold increase in k(cat) is actually more stable than wild type. This suggests that the ultimate decrease in stability may be due to random drift rather than a physical incompatibility between low temperature activity and high temperature stability. SSII shares 77. 4% identity with the naturally psychrophilic protease subtilisin S41. Although SSII and S41 differ at 85 positions, four amino acid substitutions were sufficient to generate an SSII whose low temperature activity is greater than that of S41. That none of the four are found in S41 indicates that there are multiple routes to cold adaptation.

Molecular structure and functional adaptations of hemoglobins from Antarctic marine organisms

This research dealing with fish, bird and mammal hemoglobins is in the framework of the study of the molecular basis of cold adaptation in Antarctic organisms. The study of hemoglobin structural and functional properties in Antarctic teleosts allowed a correlation of amino acid sequence, multiplicity, and oxygen‐binding features with ecological constraints. Amino acid sequences of α and β chains were analysed to build phylogenetic trees, while the organisation and expression of globin genes was studied. The respiratory proteins of Antarctic birds were investigated, in relation with the oxygen demands arising from their characteristic behaviour (diving or flight) and, in general, from the extreme conditions of the Antarctic habitat. The study of Weddell seal hemoglobins indicated that the combined effect of carbon dioxide, organic phosphates and temperature optimises oxygen delivery to all tissues in spite of their relative heterothermia. The crystallographic structure of the carbonmonoxy derivative of Trematomus newnesi major hemoglobin was resolved, giving new insight into the study of the Root effect. The molecular models of skua hemoglobins revealed the presence of a second, additional phosphate binding site located between the two α chains, paving the way to further studies.

Cryo-bioorganic chemistry: molecular interactions at low temperature.

Freezing of aqueous or organic solutions plays a pivotal role in enhancement of rate and/or yield of biomolecular reactions. The smooth conditions of the frozen state at low temperature can also suppress racemization and side-product formation of the reactions. Molecular interactions in liquid undercooled solutions, on the other hand, offer the possibility to study enzyme activity mechanisms in vitro and a chance for survival of organisms in vivo. This review illustrates the differences between frozen and liquid conditions on several small and large biomolecules, together with the synthetic use of freezing. In relation to the freezing effect on enzyme activity, a peculiar phenomenon is discussed: 'cryo-oscillations' are temporal motions of trypsin activity in frozen solution in the presence of Mn2+ ion. The molecular basis of cold adaptation is also discussed, which points to mechanisms evolved by organisms living at subzero temperatures. The factors involved in the freezing effect are shown; i.e. the role of freeze-concentration and frozen solvent surface is demonstrated and elucidated using several examples.

Psychrophilic enzymes: molecular basis of cold adaptation.

Psychrophilic organisms have successfully colonized polar and alpine regions and are able to grow efficiently at sub-zero temperatures. At the enzymatic level, such organisms have to cope with the reduction of chemical reaction rates induced by low temperatures in order to maintain adequate metabolic fluxes. Thermal compensation in cold-adapted enzymes is reached through improved turnover number and catalytic efficiency. This optimization of the catalytic parameters can originate from a highly flexible structure which provides enhanced abilities to undergo conformational changes during catalysis. Thermal instability of cold-adapted enzymes is therefore regarded as a consequence of their conformational flexibility. A survey of the psychrophilic enzymes studied so far reveals only minor alterations of the primary structure when compared to mesophilic or thermophilic homologues. However, all known structural factors and weak interactions involved in protein stability are either reduced in number or modified in order to increase their flexibility.

Trypsin and trypsinogen from an Antarctic fish: molecular basis of cold adaptation

Trypsin from Antarctic fish Paranotothenia magellanica displays molecular and kinetic properties typical of enzymes produced by psychrophilic organisms. The enzyme has a high catalytic efficiency at low and moderate temperatures and is rapidly inactivated at temperatures higher than 30°C. The nucleotide sequence was determined after mRNA extraction and cDNA synthesis. The cDNA encodes a pretrypsinogen which includes a seven residue activation peptide containing only three acidic residues preceeding the 222 amino-acid mature enzyme. A three-dimensional model of the enzyme was built. Structural parameters possibly involved in the adaptation to cold have been derived from comparison with the three-dimensional structure of the bovine enzyme. Among them are the lack of Tyr-151 in the substrate binding pocket, an overall decrease in the number of salt bridges and hydrophobicity and the increase in the surface hydrophilicity.

Molecular and biological changes in the cold-adapted "master strain" A/AA/6/60 (H2N2) influenza virus.

The live cold-adapted (ca) A/AA/6/60 influenza vaccine is being commercially developed for worldwide use in children and is being used as a model for other live vaccines. Although it has been proven safe and immunogenic, the molecular basis of cold adaptation has never been determined. To identify sequence changes responsible for cold adaptation, we have compared the sequence of the master ca vaccine strain to its progenitor wild-type virus, wt A/AA/6/60 E2 (wt2). Only 4 nt differences encoding 2 aa differences were found in three gene segments. Computer-predicted RNA folds project different secondary structures between the ca and wt2 molecules based on the two silent differences between them. Genes coding for the acidic polymerase, matrix, and nonstructural proteins are identical between the two viruses. The few differences found in the ca A/AA/6/60 virus after its long stepwise passage at 25 degrees C in primary chicken kidney cells suggest that cold adaptation resulted in greater genetic stability for the highly variable RNA genome.

Molecular adaptation of the blood of Antarctic teleosts to environmental conditions

Following the break-up of Gondwana, the drift of Antarctica to its present position and the establishment of the Antarctic Convergence, fish evolution was characterized by adaptation to progressive cooling of the environment. The decrease of erythrocyte number and haemoglobin concentration in the blood of Antarctic teleosts raises several questions concerning the physiology of respiration and the enzymatic role of erythrocytes. Our study of the molecular basis of cold adaptation includes the relationship between molecular structure and biological function of haemoglobins. Species of the suborder Notothenioidei, largely confined within the Convergence, have only one major haemoglobin, which displays the Root effect in oxygen binding; on the other hand, Zoarcidae (a family found at all latitudes) have four or five haemoglobins, only one of which displays the Root effect. In addition, our data indicate that the physiological relevance of erythrocyte-like cells, present in very small number in the blood of haemoglobinless Channichthyidae, may be linked to higher content of enzymes, such as glucose-6-phosphate dehydrogenase, in comparison with erythrocytes of red-blooded fishes.