Steffen P. Graether

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.

Quantitative and qualitative analysis of type III antifreeze protein structure and function.

Some cold water marine fishes avoid cellular damage because of freezing by expressing antifreeze proteins (AFPs) that bind to ice and inhibit its growth; one such protein is the globular type III AFP from eel pout. Despite several studies, the mechanism of ice binding remains unclear because of the difficulty in modeling the AFP-ice interaction. To further explore the mechanism, we have determined the x-ray crystallographic structure of 10 type III AFP mutants and combined that information with 7 previously determined structures to mainly analyze specific AFP-ice interactions such as hydrogen bonds. Quantitative assessment of binding was performed using a neural network with properties of the structure as input and predicted antifreeze activity as output. Using the cross-validation method, a correlation coefficient of 0.60 was obtained between measured and predicted activity, indicating successful learning and good predictive power. A large loss in the predictive power of the neural network occurred after properties related to the hydrophobic surface were left out, suggesting that van der Waal’s interactions make a significant contribution to ice binding. By combining the analysis of the neural network with antifreeze activity and x-ray crystallographic structures of the mutants, we extend the existing ice-binding model to a two-step process: 1) probing of the surface for the correct ice-binding plane by hydrogen-bonding side chains and 2) attractive van der Waal’s interactions between the other residues of the ice-binding surface and the ice, which increases the strength of the protein-ice interaction.

Modeling Pseudomonas syringae Ice-Nucleation Protein as aβ-Helical Protein

Antifreeze proteins (AFPs) inhibit the growth of ice, whereas ice-nucleation proteins (INPs) promote its formation. Although the structures of several AFPs are known, the structure of INP has been modeled thus far because of the difficulty in determining membrane protein structures. Here, we present a novel model of an INP structure from Pseudomonas syringae based on comparison with two newly determined insect AFP structures. The results suggest that both this class of AFPs and INPs may have a similar beta-helical fold and that they could interact with water through the repetitive TXT motif. By theoretical arguments, we show that the distinguishing feature between an ice inhibitor and an ice nucleator lies in the size of the ice-interacting surface. For INPs, the larger surface area acts as a template that is larger than the critical ice embryo surface area required for growth. In contrast, AFPs are small enough so that they bind to ice and inhibit further growth without acting as a nucleator.

Spruce budworm antifreeze protein: Changes in structure and dynamics at low temperature

Antifreeze proteins (AFPs) prevent the growth of ice, and are used by some organisms that live in sub-zero environments for protection against freezing. All AFPs are thought to function by an adsorption inhibition process. In order to elucidate the ice-binding mechanism, the structures of several AFPs have been determined, and have been shown to consist of different folds. Recently, the first structures of the highly active insect AFPs have been characterized. These proteins have a beta-helix structure, which adds yet another fold to the AFP family. The 90-residue spruce budworm (Choristoneura fumiferana) AFP consists of a beta-helix with 15 residues per coil. The structure contains two ranks of aligned threonine residues (known as the TXT motif), which were shown by mutagenesis experiments to be located in the ice-binding face. In our previous NMR study of this AFP at 30 degrees C, we found that the TXT face was not optimally defined because of the broadening of NMR resonances potentially due to weak oligomerization. We present here a structure of spruce budworm AFP determined at 5 degrees C, where this broadening is reduced. In addition, the 1H-15N NMR dynamics of the protein were examined at 30 degrees C and 5 degrees C. The results show that the spruce budworm AFP is more structured at 5 degrees C, and support the general observation that AFPs become more rigid as the temperature is lowered.

Freezing of a fish antifreeze protein results in amyloid fibril formation.

Amyloid is associated with a number of diseases including Alzheimers, Huntingtons, Parkinsons, and the spongiform encephalopathies. Amyloid fibrils have been formed in vitro from both disease and nondisease related proteins, but the latter requires extremes of pH, heat, or the presence of a chaotropic agent. We show, using fluorescence spectroscopy, electron microscopy, and solid-state NMR spectroscopy, that the alpha-helical type I antifreeze protein from the winter flounder forms amyloid fibrils at pH 4 and 7 upon freezing and thawing. Our results demonstrate that the freezing of some proteins may accelerate the formation of amyloid fibrils.

Effect of a mutation on the structure and dynamics of an α-helical antifreeze protein in water and ice

One strategy of psychrophilic organisms to survive subzero temperature is to produce antifreeze protein (AFPs), which inhibit the growth of macromolecular ice. To better understand the binding mechanism, the structure and dynamics of several AFPs have been studied by nuclear magnetic resonance (NMR) and X‐ray crystallography. The results have shown that different organisms can use diverse structures (α‐helix, β‐helix, or different globular folds) to achieve the same function. A number of studies have focused on understanding the relationship between the α‐helical structure of fish type I AFP and its function as an inhibitor of ice growth. The results have not explained whether the 90% activity loss caused by the conservative mutation of two threonines to serines (Thr13Ser/Thr24Ser) is attributable to a change in protein structure in solution or in ice. We examine here the structure and dynamics of the winter flounder type I AFP and the mutant Thr13Ser/Thr24Ser in both solution and solid states using a wide range of NMR approaches. Both proteins remain fully α‐helical at all temperatures and in ice, demonstrating that the activity change must therefore not be attributable to changes in the protein fold or dynamics but differences in surface properties. Proteins 2006.

Surviving winter with antifreeze proteins: Studies on budworms and beetles

Publisher Summary The rigors of cold climates have resulted in the evolution of unique, hyperactive antifreeze proteins that bind to microscopic ice crystals. Some insects, those that are freeze tolerant, upregulate metabolic pathways for the production of cryoprotectants, such as sugars or polyhydroxy alcohols. Freeze-tolerant insects raise their supercooling point, with some producing ice nucleators to ensure freezing at high subzero temperatures, and they may also accumulate other macromolecules, such as enzymes for anaerobic glycolysis. Most studied overwintering insects, have an alternative strategy for winter survival and are freeze susceptible. To avoid freezing, these insects decrease their supercooling points. They synthesize low molecular weight cryoprotectants to lower the freezing point of their hemolymph. To avoid inoculating ice, they seal themselves in cocoons or hibemacula and eliminate materials that could act as ice nucleators. Some freeze-susceptible insects may also synthesize thermal hysteresis proteins (THPs), which depress the hemolymph freezing point, relative to the melting point, by inhibiting the growth of ice until the nonequilibrium freezing point is reached. Although thermal hysteresis (TH) activity was originally described in insects, the subsequent discovery and extensive characterization of proteins with similar properties from fish, termed antifreeze proteins (AFPs), has prescribed a name change on the insect proteins. Two freeze-susceptible species, spruce budworm and mealworm beetles represent promising candidates for the purification of AFPs, because both can be reared under laboratory conditions.