Virginia K. Walker

β-Helix structure and ice-binding properties of a hyperactive antifreeze protein from an insect.

Insect antifreeze proteins (AFP) are considerably more active at inhibiting ice crystal growth than AFP from fish or plants. Several insect AFPs, also known as thermal hysteresis proteins, have been cloned and expressed. Their maximum activity is 3–4 times that of fish AFPs and they are 10–100 times more effective at micromolar concentrations. Here we report the solution structure of spruce budworm (Choristoneura fumiferana) AFP and characterize its ice-binding properties. The 9-kDa AFP is a β-helix with a triangular cross-section and rectangular sides that form stacked parallel β-sheets; a fold which is distinct from the three known fish AFP structures. The ice-binding side contains 9 of the 14 surface-accessible threonines organized in a regular array of TXT motifs that match the ice lattice on both prism and basal planes. In support of this model, ice crystal morphology and ice-etching experiments are consistent with AFP binding to both of these planes and thus may explain the greater activity of the spruce budworm antifreeze.

Hyperactive antifreeze protein from beetles

We have purified a thermal hysteresis (antifreeze) protein, with up to 100 times the specific activity of fish antifreeze proteins, from the common yellow mealworm beetle, Tenebrio molitor. It is a threonine- and cysteine-rich protein, of relative molecular mass 8,400, composed largely of 12-amino-acid repeats. We estimate that a concentration of roughly 1 mg ml−1 of this protein can account for the 5.5 °C of thermal hysteresis found in Tenebrio larvae (Fig. 1).

Freeze-Thaw Tolerance and Clues to the Winter Survival of a Soil Community

ABSTRACT Although efforts have been made to sample microorganisms from polar regions and to investigate a few of the properties that facilitate survival at freezing or subzero temperatures, soil communities that overwinter in areas exposed to alternate freezing and thawing caused by Foehn or Chinook winds have been largely overlooked. We designed and constructed a cryocycler to automatically subject soil cultures to alternating freeze-thaw cycles. After 48 freeze-thaw cycles, control Escherichia coli and Pseudomonas chlororaphis isolates were no longer viable. Mixed cultures derived from soil samples collected from a Chinook zone showed that the population complexity and viability were reduced after 48 cycles. However, when bacteria that were still viable after the freeze-thaw treatments were used to obtain selected cultures, these cultures proved to be >1,000-fold more freeze-thaw tolerant than the original consortium. Single-colony isolates obtained from survivors after an additional 48 freeze-thaw cycles were putatively identified by 16S RNA gene fragment sequencing. Five different genera were recognized, and one of the cultures, Chryseobacterium sp. strain C14, inhibited ice recrystallization, a property characteristic of antifreeze proteins that prevents the growth of large, potentially damaging ice crystals at temperatures close to the melting temperature. This strain was also notable since cell-free medium derived from cultures of it appeared to enhance the multiple freeze-thaw survival of another isolate, Enterococcus sp. strain C8. The results of this study and the development of a cryocycler should allow further investigations into the biochemical and soil community adaptations to the rigors of a Chinook environment.

Perturbation of an arctic soil microbial community by metal nanoparticles.

Technological advances allowing routine nanoparticle (NP) manufacture have enabled their use in electronic equipment, foods, clothing and medical devices. Although some NPs have antibacterial activity, little is known about their environmental impact and there is no information on the influence of NPs on soil in the possibly vulnerable ecosystems of polar regions. The potential toxicity of 0.066% silver, copper or silica NPs on a high latitude (>78°N) soil was determined using community level physiological profiles (CLPP), fatty acid methyl ester (FAME) assays and DNA analysis, including sequencing and denaturing gradient gel electrophoresis (DGGE). The results of these different investigations were amalgamated in order to develop a community toxicity indicator, which revealed that of the three NPs examined, silver NPs could be classified as highly toxic to these arctic consortia. Subsequent culture-based studies confirmed that one of the community-identified plant-associating bacteria, Bradyrhizobium canariense, appeared to have a marked sensitivity to silver NPs. Thus, NP contamination of arctic soils particularly by silver NPs is a concern and procedures for mitigation and remediation of such pollution should be a priority for investigation.

A beta-helical antifreeze protein isoform with increased activity. Structural and functional insights.

The insect spruce budworm (Choristoneura fumiferana)(Cf) produces a number of isoforms of its highly active antifreeze protein (CfAFP). Although most of the CfAFP isoforms are in the 9-kDa range, isoforms containing a 30- or 31-amino acid insertion have also been identified. Here we describe the functional and structural analysis of a selected long isoform, CfAFP-501. X-ray crystal structure determination reveals that the 31-amino acid insertion found in CfAFP-501 forms two additional loops within its highly regular β-helical structure. This effectively extends the area of the two-dimensional Thr array and ice-binding surface of the protein. The larger isoform has 3 times the thermal hysteresis activity of the 9-kDa CfAFP-337. As well, a deletion of the 31-amino acid insertion within CfAFP-501 to form CfAFP-501-Δ-2-loop, results in a protein with reduced activity similar to the shorter CfAFP isoforms. Thus, the enhanced antifreeze activity of CfAFP-501 is directly correlated to the length of its β-helical structure and hence the size of its ice-binding face.

Purification of antifreeze proteins by adsorption to ice

Antifreeze proteins (AFPs) can protect organisms from freezing injury by adsorbing to ice and inhibiting its growth. We describe here a method where ice, grown on a cold finger, is used to selectively adsorb and purify these ice-binding proteins from a crude mixture. Type III recombinant AFP was enriched approximately 50-fold after one round of partitioning into ice and purified to homogeneity by a second round. This method can also be used to purify non-ice-binding proteins by linkage to AFP domains as demonstrated by the recovery of a 50 kDa maltose-binding protein-AFP fusion from a crude lysate of Escherichia coli.

A Theoretical Model of a Plant Antifreeze Protein from Lolium perenne

Antifreeze proteins (AFPs), found in certain organisms enduring freezing environments, have the ability to inhibit damaging ice crystal growth. Recently, the repetitive primary sequence of the AFP of perennial ryegrass, Lolium perenne, was reported. This macromolecular antifreeze has high ice recrystallization inhibition activity but relatively low thermal hysteresis activity. We present here a theoretical three-dimensional model of this 118-residue plant protein based on a beta-roll domain with eight loops of 14-15 amino acids. The fold is supported by a conserved valine hydrophobic core and internal asparagine ladders at either end of the roll. Our model, which is the first proposed for a plant AFP, displays two putative, opposite-facing, ice-binding sites with surface complementarity to the prism face of ice. The juxtaposition of the two imperfect ice-binding surfaces suggests an explanation for the proteins inferior thermal hysteresis but superior ice recrystallization inhibition activity and activity when compared with fish and insect AFPs.

Antifreeze Protein from Freeze-Tolerant Grass Has a Beta-Roll Fold with an Irregularly Structured Ice-Binding Site

The grass Lolium perenne produces an ice-binding protein (LpIBP) that helps this perennial tolerate freezing by inhibiting the recrystallization of ice. Ice-binding proteins (IBPs) are also produced by freeze-avoiding organisms to halt the growth of ice and are better known as antifreeze proteins (AFPs). To examine the structural basis for the different roles of these two IBP types, we have solved the first crystal structure of a plant IBP. The 118-residue LpIBP folds as a novel left-handed beta-roll with eight 14- or 15-residue coils and is stabilized by a small hydrophobic core and two internal Asn ladders. The ice-binding site (IBS) is formed by a flat beta-sheet on one surface of the beta-roll. We show that LpIBP binds to both the basal and primary-prism planes of ice, which is the hallmark of hyperactive AFPs. However, the antifreeze activity of LpIBP is less than 10% of that measured for those hyperactive AFPs with convergently evolved beta-solenoid structures. Whereas these hyperactive AFPs have two rows of aligned Thr residues on their IBS, the equivalent arrays in LpIBP are populated by a mixture of Thr, Ser and Val with several side-chain conformations. Substitution of Ser or Val for Thr on the IBS of a hyperactive AFP reduced its antifreeze activity. LpIBP may have evolved an IBS that has low antifreeze activity to avoid damage from rapid ice growth that occurs when temperatures exceed the capacity of AFPs to block ice growth while retaining the ability to inhibit ice recrystallization.

The bugs that came in from the cold: molecular adaptations to low temperatures in insects

Abstract.The widespread distribution of insects over many ecological niches is a testimony to their evolutionary success. The colonization of environments at high latitudes or altitudes required the evolution of biochemical strategies that reduced the impact of cold or freezing stress. This review focuses on our current interests in some of the genes and proteins involved in low temperature survival in insects. Although the most widespread form of protection is the synthesis of low molecular weight polyol cryoprotectants, proteins with intrinsic protective properties, such as the thermal hysteresis or antifreeze proteins are also important. These have been cloned and characterized in certain moths and beetles. Molecular techniques allowing the isolation of genes differentially regulated by low temperatures have revealed that heat shock proteins, cold stress proteins, membrane protectants, as well as ice nucleators and other less well characterized proteins likely also play a role in cold hardiness.

Cold tolerance and proline metabolic gene expression in Drosophila melanogaster

Treatment of Drosophila melanogaster adults with an inhibitor of protein synthesis led to a decrease in intrinsic cold-shock tolerance, but no difference in the rapid cold hardening response, which is apparent only if a period at 4 degrees C precedes the cold stress. Increases in energy reserves, including proline, were found in lines of flies selected for resistance to chilling injury. Since an increase in proline levels has been associated with overwintering in insects, and for salt and cold tolerance in plants, an RNase protection assay was developed to assess changes in transcript abundance for two genes encoding enzymes important for proline metabolism, pyrroline 5-carboxylate reductase and proline oxidase. The mRNA levels did not change in response to low temperature, but the high level of pyrroline 5-carboxylate reductase transcript is consistent with the interpretation that a large proline pool is important for Drosophila metabolism and survival during cold stress.