Ice-binding proteins and bioinspired synthetic mimics in non-physiological environments.

Ice-binding proteins (IBPs) are produced by a variety of organisms to prevent internal damage caused by ice crystal growth. Synthetic analogs are being designed to mimic beneficial properties of IBPs while mitigating drawbacks related to the use of biological proteins. While a multitude of engineering applications could benefit from the inhibition and control of ice formation and growth, synthetic analogs tend to be less potent than biological IBPs, and both IBPs and synthetic analogs tend to exhibit lower performance in non-physiological (i.e., non-biological) solutions. This review examines the ice interaction properties and performance of IBPs and their synthetic analogs in non-physiological environments. Common methods to measure ice interactions are discussed (i.e., thermal hysteresis, ice recrystallization inhibition, ice growth rate, and ice nucleation). A quantitative meta-analysis of material performance in non-physiological environments is presented, along with a discussion of future research directions. The findings presented herein can inform IBP and synthetic mimic selection to control ice interactions in a wide variety of materials science and engineering applications, including cell, tissue, and organ cryopreservation, food storage and transport, freeze-thaw damage of cementitious materials, and anti-icing surfaces for aerospace vehicles, solar panels, and wind turbines.

Bioinspired Threonine-Based Polymers with Potent Ice Recrystallization Inhibition Activity.

Ice growth mitigation is a pervasive challenge for multiple industries. In nature, ice-binding proteins (IBPs) demonstrate potent ice growth prevention through ice recrystallization inhibition (IRI). However, IBPs are expensive, difficult to produce in large quantities, and exhibit minimal resilience to nonphysiological environmental stressors, such as pH. For these reasons, researchers have turned to bioinspired polymeric materials that mimic IBP behavior. To date, however, no synthetic polymer has rivaled the ability of native IBPs to display IRI activity at ultralow nanomolar concentrations. In this work, we study the IRI activity of peptides and polypeptides inspired by common ice-binding residues of IBPs to inform the synthesis and characterization of a potent bioinspired polymer that mimics IBP behavior. We show first that the threonine polypeptide (pThr) displays the best IRI activity in phosphate-buffered saline (PBS). Second, we use pThr as a molecular model to synthesize and test a bioinspired polymer, poly(2-hydroxypropyl methacrylamide) (pHPMA). We show that pHPMA exhibits potent IRI activity in neutral PBS at ultralow concentrations (0.01 mg/mL). pHPMA demonstrates potent IRI activity at low molecular weights (2.3 kDa), with improved activity at higher molecular weights (32.8 kDa). These results substantiate that pHPMA is a robust molecule that mitigates ice crystal growth at concentrations similar to native IBPs.

Mechanical evaluation of 3D printed biomimetic non-Euclidean saddle geometries mimicking the mantis shrimp.

Engineering design has drawn inspiration from naturally occurring structures to advance manufacturing processes and products, termed biomimetics. For example, the mantis shrimp, orderStomatopoda, is capable of producing one of the fastest appendage strikes in the world with marginal musculoskeletal displacement. The extreme speed of the mantis shrimp's raptorial appendage is due to the non-Euclidean hyperbolic paraboloid (i.e. saddle) shape within the dorsal region of the merus, which allows substantial energy storage through compression in the sagittal plane. Here, investigation of 3D printed synthetic geometries inspired by the mantis shrimp saddle geometry has revealed insights for elastic energy storage (i.e. spring-like) applications. Saddles composed of either astiffor aflexibleresin were investigated for spring response to explore the geometric effects. By modulating the saddle geometry and testing the spring response, it was found that, for thestiffresin, the spring constant was improved as the curvature of the contact and orthogonal faces were maximized and minimized, respectively. For theflexibleresin, it was found that the spring constant increased by less than 250 N mm-1as the saddle geometry changed, substantiating that the flexible component of mantis saddles does not contribute to energy storage capabilities. The geometries of two saddles from the mantis shrimp speciesO. scyllaruswere estimated and exhibited similar trends to manufactured saddles, suggesting that modulating saddle geometry can be used for tailored energy storage moduli in spatially constrained engineering applications.

Effect of pH on the activity of ice-binding protein from Marinomonas primoryensis.

The ability of an ice-binding protein (IBP) from Marinomonas primoryensis (MpIBP) to influence ice crystal growth and structure in nonphysiological pH environments was investigated in this work. The ability for MpIBP to retain ice interactivity under stressed environmental conditions was determined via (1) a modified splat assay to determine ice recrystallization inhibition (IRI) of polycrystalline ice and (2) nanoliter osmometry to evaluate the ability of MpIBP to dynamically shape the morphology of a single ice crystal. Circular dichroism (CD) was used to relate the IRI and DIS activity of MpIBP to secondary structure. The results illustrate that MpIBP secondary structure was stable between pH 6 and pH 10. It was found that MpIBP did not interact with ice at pH ≤ 4 or pH ≥ 13. At 6 ≤ pH ≥ 12 MpIBP exhibited a reduction in grain size of ice crystals as compared to control solutions and demonstrated dynamic ice shaping at 6 ≤ pH ≥ 10. The results substantiate that MpIBP retains some secondary structure and function in non-neutral pH environments; thereby, enabling its potential utility in nonphysiological materials science and engineering applications.

Ice-Binding Protein from Shewanella frigidimarinas Inhibits Ice Crystal Growth in Highly Alkaline Solutions.

The ability of a natural ice-binding protein from Shewanella frigidimarina (SfIBP) to inhibit ice crystal growth in highly alkaline solutions with increasing pH and ionic strength was investigated in this work. The purity of isolated SfIBP was first confirmed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion chromatography with an ultraviolet detector (SEC-UV). Protein stability was evaluated in the alkaline solutions using circular dichroism spectroscopy, SEC-UV, and SDS-PAGE. SfIBP ice recrystallization inhibition (IRI) activity, a measure of ice crystal growth inhibition, was assessed using a modified splat assay. Statistical analysis of results substantiated that, despite partial denaturation and misfolding, SfIBP limited ice crystal growth in alkaline solutions (pH ≤ 12.7) with ionic strength I ≤ 0.05 mol/L, but did not exhibit IRI activity in alkaline solutions where pH ≥ 13.2 and I ≥ 0.16 mol/L. IRI activity of SfIBP in solutions with pH ≤ 12.7 and I ≤ 0.05 mol/L demonstrated up to ≈ 66% reduction in ice crystal size compared to neat solutions.