Influence of polymer chain architecture of poly(vinyl alcohol) on the inhibition of ice recrystallization

\u3cp\u3ePoly(vinyl alcohol) (PVA) is a water-soluble synthetic polymer well-known to effectively block the recrystallization of ice. The effect of polymer chain architecture on the ice recrystallization inhibition (IRI) by PVA remains unexplored. In this work, the synthesis of PVA molecular bottlebrushes is described via a combination of atom-transfer radical polymerization and reversible addition-fragmentation chain-transfer polymerization. The facile preparation of the PVA bottlebrushes is performed via the selective hydrolysis of the chloroacetate esters of the poly(vinyl chloroacetate) (PVClAc) side chains of a PVClAc precursor bottlebrush. The IRI efficacy of the PVA bottlebrush is quantitatively compared to linear PVA. The results show that even if the PVA chains are densely grafted onto a rigid polymer backbone, the IRI activity of PVA is maintained, demonstrating the flexibility in PVA polymer chain architecture for the design of synthetic PVA-based ice growth inhibitors.\u3c/p\u3

Interaction of ice binding proteins with ice, water and ions

\u3cp\u3eIce binding proteins (IBPs) are produced by various cold-adapted organisms to protect their body tissues against freeze damage. First discovered in Antarctic fish living in shallow waters, IBPs were later found in insects, microorganisms, and plants. Despite great structural diversity, all IBPs adhere to growing ice crystals, which is essential for their extensive repertoire of biological functions. Some IBPs maintain liquid inclusions within ice or inhibit recrystallization of ice, while other types suppress freezing by blocking further ice growth. In contrast, ice nucleating proteins stimulate ice nucleation just below 0 °C. Despite huge commercial interest and major scientific breakthroughs, the precise working mechanism of IBPs has not yet been unraveled. In this review, the authors outline the state-of-the-art in experimental and theoretical IBP research and discuss future scientific challenges. The interaction of IBPs with ice, water and ions is examined, focusing in particular on ice growth inhibition mechanisms.\u3c/p\u3

Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins

\u3cp\u3eAntifreeze proteins (AFP\u3csub\u3es\u3c/sub\u3e) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFP\u3csub\u3es\u3c/sub\u3e have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of commonly used colligative antifreezes such as salts and alcohols, the advantage of using AFP\u3csub\u3es\u3c/sub\u3e as an additive is that they do not alter the physicochemical properties of the water-based material. Here, we report the first comprehensive evaluation of thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity of all major classes of AFP\u3csub\u3es\u3c/sub\u3e using cryoscopy, sonocrystallization, and recrystallization assays. The results show that TH activities determined by cryoscopy and sonocrystallization differ markedly, and that TH and IRI activities are not correlated. The absence of a distinct correlation in antifreeze activity points to a mechanistic difference in ice growth inhibition by the different classes of AFP\u3csub\u3es\u3c/sub\u3e: blocking fast ice growth requires rapid nonbasal plane adsorption, whereas basal plane adsorption is only relevant at long annealing times and at small undercooling. These findings clearly demonstrate that biomimetic analogs of antifreeze (glyco)proteins should be tailored to the specific requirements of the targeted application.\u3c/p\u3

Ca2+-stabilized adhesin helps an Antarctic bacterium reach out and bind ice

\u3cp\u3eThe large size of a 1.5-MDa ice-binding adhesin [MpAFP (Marinomonas primoryensis antifreeze protein)] from an Antarctic Gram-negative bacterium, M. primoryensis, is mainly due to its highly repetitive RII (Region II). MpAFP-RII contains roughly 120 tandem copies of an identical 104-residue repeat. We have previously determined that a single RII repeat folds as a Ca\u3csup\u3e2+\u3c/sup\u3e- dependent immunoglobulin-like domain. Here, we solved the crystal structure of RII tetra-tandemer (four tandem RII repeats) to a resolution of 1.8 Å. The RII tetra-tandemer reveals an extended (∼190-Å x ∼25-Å), rod-like structure with four RII-repeats aligned in series with each other. The inter-repeat regions of the RII tetra-tandemer are strengthened by Ca \u3csup\u3e2+\u3c/sup\u3e bound to acidic residues. SAXS (small-angle X-ray scattering) profiles indicate the RII tetra-tandemer is significantly rigidified upon Ca\u3csup\u3e2+\u3c/sup\u3e binding, and that the protein's solution structure is in excellent agreement with its crystal structure. We hypothesize that >600 Ca\u3csup\u3e2+\u3c/sup\u3e help rigidify the chain of ∼120 104-residue repeats to form a ∼0.6 μm rod-like structure in order to project the ice-binding domain of MpAFP away from the bacterial cell surface. The proposed extender role of RII can help the strictly aerobic, motile bacterium bind ice in the upper reaches of the Antarctic lake where oxygen and nutrients are most abundant. Ca \u3csup\u3e2+\u3c/sup\u3e-induced rigidity of tandem Ig-like repeats in large adhesins might be a general mechanism used by bacteria to bind to their substrates and help colonize specific niches.\u3c/p\u3

Observation of ice-like water layers at an aqueous protein surface

\u3cp\u3eWe study the properties of water at the surface of an antifreeze protein with femtosecond surface sum frequency generation spectroscopy. We find clear evidence for the presence of ice-like water layers at the ice-binding site of the protein in aqueous solution at temperatures above the freezing point. Decreasing the temperature to the biological working temperature of the protein (0°C to -2°C) increases the amount of ice-like water, while a single point mutation in the ice-binding site is observed to completely disrupt the ice-like character and to eliminate antifreeze activity. Our observations indicate that not the protein itself but ordered ice-like water layers are responsible for the recognition and binding to ice.\u3c/p\u3

Reversible blocking of antibodies using bivalent peptide-DNA conjugates allows protease-activatable targeting

Antibody-based molecular recognition plays a dominant role in the life sciences ranging from applications in diagnostics and molecular imaging to targeted drug delivery and therapy. Here we report a generic approach to introduce protease sensitivity into antibody-based targeting by taking advantage of the intrinsic ability of antibodies to engage in multivalent interactions. Bivalent peptide ligands with dsDNA as a rigid linker were shown to effectively bridge the relatively large distance between the two antigen binding sites within the same antibody, yielding exclusively the cyclic 1:1 antibody–ligand complex. Size exclusion chromatography and small angle X-scattering were used to study the types of complexes formed between a model antibody and peptide–dsDNA conjugates displaying 1 or 2 peptide ligands and different linker lengths. Competitive binding assays using fluorescence anisotropy revealed that the interaction between bivalent peptide–dsDNA conjugate and antibody is 500-fold stronger than that of the monovalent peptide, allowing effective blocking of the antigen binding sites in a non-covalent manner. Cleavage of the linker between the peptide epitope and the DNA by matrix metalloprotease 2 disables this strong bivalent interaction and was shown to effectively restore the binding activity of the antibody in an in vitro binding assay. The approach presented here is broadly applicable, because it takes advantage of the Y-shaped multivalent presentation of antigen binding sites common to all antibodies and could be extended to control antibody activity by other input signals

Structure of a 1.5-MDa adhesin that binds its antarctic bacterium to diatoms and ice

Bacterial adhesins are modular cell-surface proteins that mediate adherence to other cells, surfaces, and ligands. The Antarctic bacterium Marinomonas primoryensis uses a 1.5-MDa adhesin comprising over 130 domains to position it on ice at the top of the water column for better access to oxygen and nutrients. We have reconstructed this 0.6-μm-long adhesin using a “dissect and build” structural biology approach and have established complementary roles for its five distinct regions. Domains in region I (RI) tether the adhesin to the type I secretion machinery in the periplasm of the bacterium and pass it through the outer membrane. RII comprises ~120 identical immunoglobulin-like β-sandwich domains that rigidify on binding Ca2+ to project the adhesion regions RIII and RIV into the medium. RIII contains ligand-binding domains that join diatoms and bacteria together in a mixed-species community on the underside of sea ice where incident light is maximal. RIV is the ice-binding domain, and the terminal RV domain contains several “repeats-in-toxin” motifs and a noncleavable signal sequence that target proteins for export via the type I secretion system. Similar structural architecture is present in the adhesins of many pathogenic bacteria and provides a guide to finding and blocking binding domains to weaken infectivity

Interaction of antifreeze proteins with water

Antifreeze proteins and antifreeze glycoproteins (AF(G)Ps) enable the survival of various cold-adapted organisms in freezing and subfreezing habitats by preventing the macroscopic growth of ice crystals. Regardless of their great structural diversity are all AF(G)Ps capable to adhere to growing ice crystals, a quality that is essential for their biological functions. Despite commercial interest and significant scientific breakthroughs has the precise working mechanism of antifreeze proteins not yet been unraveled. In this chapter we highlight the latest state-of-the art experimental and theoretical antifreeze protein research on the solution behavior of AF(G)Ps and their interaction with the solvent. Protein-water interactions are of general interest owing to the importance of protein hydration for the structure, stability, and activity of almost all proteins. We focus in particular on the direct interaction of AF(G)Ps with water and its role in the working mechanism of these unique proteins