Mechanism of bacterial adaptation to low temperature.
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Survival of bacteria at low temperatures provokes scientific interest because of several reasons. Investigations in this area promise insight into one of the mysteries of life science - namely, how the machinery of life operates at extreme environments. Knowledge obtained from these studies is likely to be useful in controlling pathogenic bacteria, which survive and thrive in cold-stored food materials. The outcome of these studies may also help us to explore the possibilities of existence of life in distant frozen planets and their satellites. (+info)
Insight into the binding of antifreeze proteins to ice surfaces via 13C spin lattice relaxation solid-state NMR.
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The primary sequences of type I antifreeze proteins (AFPs) are Ala rich and contain three 11-residue repeat units beginning with threonine residues. Their secondary structures consist of alpha-helices. Previous activity study of side-chain mutated AFPs suggests that the ice-binding side of type I AFPs comprises the Thr side chains and the conserved i + 4 and i + 8 Ala residues, where i indicates the positions of the Thrs. To find structural evidence for the AFP's ice-binding side, a variable-temperature dependent (13)C spin lattice relaxation solid-state NMR experiment was carried out for two Ala side chain (13)C labeled HPLC6 isoforms of the type I AFPs each frozen in H(2)O and D(2)O, respectively. The first one was labeled on the equivalent 17th and 21st Ala side chains (i + 4, 8), and the second one on the equivalent 8th, 19th, and 30th Ala side chains (i + 6). The two kinds of labels are on the opposite sides of the alpha-helical AFP. A model of Ala methyl group rotation/three-site rotational jump combined with water molecular reorientation was tested to probe the interactions of the methyl groups with the proximate water molecules. Analysis of the T(1) data shows that there could be 10 water molecules closely capping an i + 4 or an i + 8 methyl group within the range of van der Waals interaction, whereas the surrounding water molecules to the i + 6 methyl groups could be looser. This study suggests that the side of the alpha-helical AFP comprising the i + 4 and i + 8 Ala methyl groups could interact with the ice surface in the ice/water interface. (+info)
Wheat EST resources for functional genomics of abiotic stress.
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BACKGROUND: Wheat is an excellent species to study freezing tolerance and other abiotic stresses. However, the sequence of the wheat genome has not been completely characterized due to its complexity and large size. To circumvent this obstacle and identify genes involved in cold acclimation and associated stresses, a large scale EST sequencing approach was undertaken by the Functional Genomics of Abiotic Stress (FGAS) project. RESULTS: We generated 73,521 quality-filtered ESTs from eleven cDNA libraries constructed from wheat plants exposed to various abiotic stresses and at different developmental stages. In addition, 196,041 ESTs for which tracefiles were available from the National Science Foundation wheat EST sequencing program and DuPont were also quality-filtered and used in the analysis. Clustering of the combined ESTs with d2_cluster and TGICL yielded a few large clusters containing several thousand ESTs that were refractory to routine clustering techniques. To resolve this problem, the sequence proximity and "bridges" were identified by an e-value distance graph to manually break clusters into smaller groups. Assembly of the resolved ESTs generated a 75,488 unique sequence set (31,580 contigs and 43,908 singletons/singlets). Digital expression analyses indicated that the FGAS dataset is enriched in stress-regulated genes compared to the other public datasets. Over 43% of the unique sequence set was annotated and classified into functional categories according to Gene Ontology. CONCLUSION: We have annotated 29,556 different sequences, an almost 5-fold increase in annotated sequences compared to the available wheat public databases. Digital expression analysis combined with gene annotation helped in the identification of several pathways associated with abiotic stress. The genomic resources and knowledge developed by this project will contribute to a better understanding of the different mechanisms that govern stress tolerance in wheat and other cereals. (+info)
Nonhepatic origin of notothenioid antifreeze reveals pancreatic synthesis as common mechanism in polar fish freezing avoidance.
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Phylogenetically diverse polar and subpolar marine teleost fishes have evolved antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) to avoid inoculative freezing by internalized ice. For over three decades since the first fish antifreeze (AF) protein was discovered, many studies of teleost freezing avoidance showed hepatic AF synthesis and distribution within the circulation as pivotal in preventing the blood, and therefore the fish, from freezing. We have uncovered an important twist to this long-held paradigm: the complete absence of liver synthesis of AFGPs in any life stage of the Antarctic notothenioids, indicating that the liver plays no role in the freezing avoidance in these fishes. Instead, we found the exocrine pancreas to be the major site of AFGP synthesis and secretion in all life stages, and that pancreatic AFGPs enter the intestinal lumen via the pancreatic duct to prevent ingested ice from nucleating the hyposmotic intestinal fluids. AFGPs appear to remain undegraded in the intestinal milieu, and the composition and relative abundance of intestinal AFGP isoforms are nearly identical to serum AFGPs. Thus, the reabsorption of intact pancreas-derived intestinal AFGPs, and not the liver, is the likely source of circulatory AFGPs in notothenioid fishes. We examined diverse northern fish taxa and Antarctic eelpouts with hepatic synthesis of bloodborne AF and found that they also express secreted pancreatic AF of their respective types. The evolutionary convergence of this functional physiology underscores the hitherto largely unrecognized importance of intestinal freezing prevention in polar teleost freezing avoidance, especially in the chronically icy Antarctic waters. (+info)
Structural modeling of snow flea antifreeze protein.
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The glycine-rich antifreeze protein recently discovered in snow fleas exhibits strong freezing point depression activity without significantly changing the melting point of its solution (thermal hysteresis). BLAST searches did not detect any protein with significant similarity in current databases. Based on its circular dichroism spectrum, discontinuities in its tripeptide repeat pattern, and intramolecular disulfide bonding, a detailed theoretical model is proposed for the 6.5-kDa isoform. In the model, the 81-residue protein is organized into a bundle of six short polyproline type II helices connected (with one exception) by proline-containing turns. This structure forms two sheets of three parallel helices, oriented antiparallel to each other. The central helices are particularly rich in glycines that facilitate backbone carbonyl-amide hydrogen bonding to four neighboring helices. The modeled structure has similarities to polyglycine II proposed by Crick and Rich in 1955 and is a close match to the polyproline type II antiparallel sheet structure determined by Traub in 1969 for (Pro-Gly-Gly)(n). Whereas the latter two structures are formed by intermolecular interactions, the snow flea antifreeze is stabilized by intramolecular interactions between the helices facilitated by the regularly spaced turns and disulfide bonds. Like several other antifreeze proteins, this modeled protein is amphipathic with a putative hydrophobic ice-binding face. (+info)
Protein interaction with ice.
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Many organisms have evolved novel mechanisms to minimize freezing injury due to extracellular ice formation. This article reviews our present knowledge on the structure and mode of action of two types of proteins capable of ice interaction. The antifreeze proteins inhibit ice crystal formation and alter ice growth habits. The ice nucleation proteins, on the other hand, provide a proper template to stimulate ice growth. The potential applications of these proteins in different industries are discussed. (+info)
Fluorescence microscopy evidence for quasi-permanent attachment of antifreeze proteins to ice surfaces.
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Many organisms are protected from freezing by the presence of extracellular antifreeze proteins (AFPs), which bind to ice, modify its morphology, and prevent its further growth. These proteins have a wide range of applications including cryopreservation, frost protection, and as models in biomineralization research. However, understanding their mechanism of action remains an outstanding challenge. While the prevailing adsorption-inhibition hypothesis argues that AFPs must bind irreversibly to ice to arrest its growth, other theories suggest that there is exchange between the bound surface proteins and the free proteins in solution. By conjugating green fluorescence protein (GFP) to a fish AFP (Type III), we observed the binding of the AFP to ice. This was accomplished by monitoring the presence of GFP-AFP on the surface of ice crystals several microns in diameter using fluorescence microscopy. The lack of recovery of fluorescence after photobleaching of the GFP component of the surface-bound GFP-AFP shows that there is no equilibrium surface-solution exchange of GFP-AFP and thus supports the adsorption-inhibition mechanism for this type of AFP. Moreover, our study establishes the utility of fluorescently labeled AFPs as a research tool for investigating the mechanisms underlying the activity of this diverse group of proteins. (+info)
Antifreeze proteins at the ice/water interface: three calculated discriminating properties for orientation of type I proteins.
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Antifreeze proteins (AFPs) protect many plants and organisms from freezing in low temperatures. Of the different AFPs, the most studied AFP Type I from winter flounder is used in the current computational studies to gain molecular insight into its adsorption at the ice/water interface. Employing molecular dynamics simulations, we calculate the free energy difference between the hydrophilic and hydrophobic faces of the protein interacting with ice. Furthermore, we identify three properties of Type I "antifreeze" proteins that discriminate among these two orientations of the protein at the ice/water interface. The three properties are: the "surface area" of the protein; a measure of the interaction of the protein with neighboring water molecules as determined by the number of hydrogen bond count, for example; and the side-chain orientation angles of the threonine residues. All three discriminants are consistent with our free energy results, which clearly show that the hydrophilic protein face orientations toward the ice/water interface, as hypothesized from experimental and ice/vacuum simulations, are incorrect and support the hypothesis that the hydrophobic face is oriented toward the ice/water interface. The adsorption free energy is calculated to be 2-3 kJ/mol. (+info)