An Ultrapotent, Ultraeconomical, Antifreeze Polypeptide.

The growth of large ice crystals during freeze and thaw events is a challenge in diverse settings from transportation and agriculture to foods and biomedicine. Design, synthesis, and evaluation of antifreeze polypeptides that inhibit ice crystal growth at µg concentrations are reported herein. The polypeptides, composed of Ala and Glu, are prepared using economical methodology, are stable after thermal events, are biodegradable, and are nontoxic to human cells.

Optimization of the Gold Nanoparticle Colorimetric Assay for Screening and Quantifying Ice Recrystallization Inhibition Activity of Antifreeze Proteins.

Antifreeze proteins or ice-binding proteins can be found in a variety of organisms, including fish, insects, and plants. These proteins are responsible for hindering ice crystal growth, a process known as ice recrystallization inhibition (IRI), allowing the organism to survive in subzero temperatures. Antifreeze proteins are an attractive area of research because of their potential applications in wide-ranging areas, such as food and crops preservation, industry, and cryopreservation.

Polyol-Induced 100-Fold Enhancement of Bacterial Ice Nucleation Efficiency.

Ice-nucleating proteins (INPs) from bacteria like are among the most effective ice nucleators known. However, large INP aggregates with maximum ice nucleation activity (at approximately -2 °C) typically account for less than 1% of the overall ice nucleation activity in bacterial samples. This study demonstrates that polyols significantly enhance the assembly of INPs into large aggregates, dramatically improving bacterial ice nucleation efficiency.

Hierarchical assembly and environmental enhancement of bacterial ice nucleators.

Bacterial ice nucleating proteins (INPs) are exceptionally effective in promoting the kinetically hindered transition of water to ice. Their efficiency relies on the assembly of INPs into large functional aggregates, with the size of ice nucleation sites determining activity. Experimental freezing spectra have revealed two distinct, defined aggregate sizes, typically classified as class A and C ice nucleators (INs).

Simple Method to Assess Foam Structure and Stability using Hydrophobin and BSA as Model Systems.

The properties and arrangement of surface-active molecules at air-water interfaces influence foam stability and bubble shape. Such multiscale-relationships necessitate a well-conducted analysis of mesoscopic foam properties. We introduce a novel automated and precise method to characterize bubble growth, size distribution and shape based on image analysis and using the machine learning algorithm Cellpose.

Functional aggregation of cell-free proteins enables fungal ice nucleation.

Biological ice nucleation plays a key role in the survival of cold-adapted organisms. Several species of bacteria, fungi, and insects produce ice nucleators (INs) that enable ice formation at temperatures above -10 °C. Bacteria and fungi produce particularly potent INs that can promote water crystallization above -5 °C.

Measurement of Ice Nucleation Activity of Biological Samples.

Experimentation with ice-nucleating biomolecules is needed to advance the fundamental understanding of biotic heterogeneous ice nucleation. Standard experimental procedures vary with sample type. Here we describe a generalized primary purification and analysis process to measure ice nucleation activity of biological samples using an advanced freezing droplet assay.

True Origin of Amide I Shifts Observed in Protein Spectra Obtained with Sum Frequency Generation Spectroscopy.

Accurate determination of protein structure at interfaces is critical for understanding protein interactions, which is directly relevant to a molecular-level understanding of interfacial proteins in biology and medicine. Vibrational sum frequency generation (VSFG) spectroscopy is often used for probing the protein amide I mode, which reports protein structures at interfaces. Observed peak shifts are attributed to conformational changes and often form the foundation of hypotheses explaining protein working mechanisms.

Lichen species across Alaska produce highly active and stable ice nucleators.

Forty years ago, lichens were identified as extraordinary biological ice nucleators (INs) that enable ice formation at temperatures close to 0°C. By employing INs, lichens thrive in freezing environments that surpass the physiological limits of other vegetation, thus making them the majority of vegetative biomass in northern ecosystems. Aerosolized lichen INs might further impact cloud glaciation and have the potential to alter atmospheric processes in a warming Arctic.

AlphaFold2 models indicate that protein sequence determines both structure and dynamics.

AlphaFold 2 (AF2) has placed Molecular Biology in a new era where we can visualize, analyze and interpret the structures and functions of all proteins solely from their primary sequences. We performed AF2 structure predictions for various protein systems, including globular proteins, a multi-domain protein, an intrinsically disordered protein (IDP), a randomized protein, two larger proteins (> 1000 AA), a heterodimer and a homodimer protein complex. Our results show that along with the three dimensional (3D) structures, AF2 also decodes protein sequences into residue flexibilities via both the predicted local distance difference test (pLDDT) scores of the models, and the predicted aligned error (PAE) maps.

Toward Understanding Bacterial Ice Nucleation.

Bacterial ice nucleators (INs) are among the most effective ice nucleators known and are relevant for freezing processes in agriculture, the atmosphere, and the biosphere. Their ability to facilitate ice formation is due to specialized ice-nucleating proteins (INPs) anchored to the outer bacterial cell membrane, enabling the crystallization of water at temperatures up to -2 °C. In this Perspective, we highlight the importance of functional aggregation of INPs for the exceptionally high ice nucleation activity of bacterial ice nucleators.

Ice Recrystallization Inhibition Is Insufficient to Explain Cryopreservation Abilities of Antifreeze Proteins.

Antifreeze proteins (AFPs) and glycoproteins (AFGPs) are exemplary at modifying ice crystal growth and at inhibiting ice recrystallization (IRI) in frozen solutions. These properties make them highly attractive for cold storage and cryopreservation applications of biological tissue, food, and other water-based materials. The specific requirements for optimal cryostorage remain unknown, but high IRI activity has been proposed to be crucial.

Cryofouling avoidance in the Antarctic scallop Adamussium colbecki.

The presence of supercooled water in polar regions causes anchor ice to grow on submerged objects, generating costly problems for engineered materials and life-endangering risks for benthic communities. The factors driving underwater ice accretion are poorly understood, and passive prevention mechanisms remain unknown. Here we report that the Antarctic scallop Adamussium colbecki appears to remain ice-free in shallow Antarctic marine environments where underwater ice growth is prevalent.

pH effects on the molecular structure and charging state of β-Escin biosurfactants at the air-water interface.

Saponins like β-escin exhibit an unusually high surface activity paired with a remarkable surface rheology which makes them as biosurfactants highly interesting for applications in soft matter colloids and at interfaces. We have applied vibrational sum-frequency generation (SFG) to study β-escin adsorption layers at the air-water interface as a function of electrolyte pH and compare the results from SFG spectroscopy to complementary experiments that have addressed the surface tension and the surface dilational rheology. SFG spectra of β-escin modified air-water interfaces demonstrate that the SFG intensity of OH stretching vibrations from interfacial water molecules is a function of pH and dramatically increases when the pH is increased from acidic to basic conditions and reaches a plateau at a solution pH of > 6.

Disaccharide Residues are Required for Native Antifreeze Glycoprotein Activity.

Antifreeze glycoproteins (AFGPs) are able to bind to ice, halt its growth, and are the most potent inhibitors of ice recrystallization known. The structural basis for AFGP's unique properties remains largely elusive. Here we determined the antifreeze activities of AFGP variants that we constructed by chemically modifying the hydroxyl groups of the disaccharide of natural AFGPs.

Ice Nucleation Activity of Perfluorinated Organic Acids.

Perfluorinated acids (PFAs) are widely used synthetic chemical compounds, highly resistant to environmental degradation. The widespread PFA contamination in remote regions such as the High Arctic implies currently not understood long-range atmospheric transport pathways. Here, we report that perfluorooctanoic acid (PFOA) initiates heterogeneous ice nucleation at temperatures as high as -16 °C.

Specific Ion-Protein Interactions Influence Bacterial Ice Nucleation.

Ice nucleation-active bacteria are the most efficient ice nucleators known, enabling the crystallization of water at temperatures close to 0 °C, thereby overcoming the kinetically hindered phase transition process at these conditions. Using highly specialized ice-nucleating proteins (INPs), they can cause frost damage to plants and influence the formation of clouds and precipitation in the atmosphere. In nature, the bacteria are usually found in aqueous environments containing ions.

Interfacial Water Ordering Is Insufficient to Explain Ice-Nucleating Protein Activity.

Ice-nucleating proteins (INPs) found in bacteria are the most effective ice nucleators known, enabling the crystallization of water at temperatures close to 0 °C. Although their function has been known for decades, the underlying mechanism is still under debate. Here, we show that INPs from in aqueous solution exhibit a defined solution structure and show no significant conformational changes upon cooling.

Inhibition of Bacterial Ice Nucleators Is Not an Intrinsic Property of Antifreeze Proteins.

Cold-adapted organisms use antifreeze proteins (AFPs) or ice-nucleating proteins (INPs) for the survival in freezing habitats. AFPs have been reported to be able to inhibit the activity of INPs, a property that would be of great physiological relevance. The generality of this effect is not understood, and for the few known examples of INP inhibition by AFPs, the molecular mechanisms remain unclear.

Freezing of Aqueous Carboxylic Acid Solutions on Ice.

We study the properties of acetic acid and propionic acid solutions at the surface of monocrystalline ice with surface-specific vibrational sum-frequency generation (VSFG) and heterodyne-detected vibrational sum-frequency generation spectroscopy (HD-VSFG). When we decrease the temperature toward the eutectic point of the acid solutions, we observe the formation of a freeze concentrated solution (FCS) of the carboxylic acids that is brought about by a freeze-induced phase separation (FIPS). The freeze concentrated solution freezes on top of the ice surface as we cool the system below the eutectic point.

Decoding the molecular water structure at complex interfaces through surface-specific spectroscopy of the water bending mode.

The structure of interfacial water determines atmospheric chemistry, wetting properties of materials, and protein folding. The challenge of investigating the properties of specific interfacial water molecules has frequently been confronted using surface-specific sum-frequency generation (SFG) vibrational spectroscopy using the O-H stretch mode. While perfectly suited for the water-air interface, for complex interfaces, a potential complication arises from the contribution of hydroxyl or amine groups of non-water species present at the surface, such as surface hydroxyls on minerals, or O-H and N-H groups contained in proteins.

Synergy between Antifreeze Proteins Is Driven by Complementary Ice-Binding.

In some cold-adapted organisms, over a dozen isoforms of antifreeze (glyco)proteins or AF(G)Ps are present. Although these isoforms are structurally similar, their ability to inhibit ice growth varies significantly, and, in some fish, passive isoforms can be much more abundant than the active ones. Laboratory experiments demonstrated more than a decade ago that mixtures of AFP isoforms can exhibit synergistic enhancement of each other's activity.

Effect of Antifreeze Glycoproteins on Organoid Survival during and after Hypothermic Storage.

We study the effect of antifreeze glycoproteins (AFGPs) on the survival of organoids under hypothermic conditions. We find that the survival of organoids in cold conditions depends on their developmental stage. Mature organoids die within 24 h when being stored at 4 °C, while cystic organoids can survive up to 48 h.

Determination of the Solution Structure of Antifreeze Glycoproteins Using Two-Dimensional Infrared Spectroscopy.

We study the solution structure of antifreeze glycoproteins (AFGPs) with linear and two-dimensional infrared spectroscopy (2D-IR). With 2D-IR, we study the coupling between the amide I and amide II vibrations of AFGPs. The measured nonlinear spectral response constitutes a much more clearly resolved amide I spectrum than the linear absorption spectrum of the amide I vibrations and allows us to identify the different structural elements of AFGPs in solution.

Antifreeze Glycoproteins Bind Irreversibly to Ice.

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) inhibit ice growth via an adsorption-inhibition mechanism that assumes irreversible binding of AF(G)Ps to embryonic ice crystals and the inhibition of further growth. The irreversible binding of antifreeze glycoproteins (AFGPs) to ice has been questioned and remains poorly understood. Here, we used microfluidics and fluorescence microscopy to investigate the nature of the binding of small and large AFGP isoforms.