Ice Recrystallization Inhibition of Ice-Binding Proteins and Bioinspired Synthetic Mimics In Extreme Environments

Delesky, Elizabeth Anna

Graduate Thesis Or Dissertation

Ice Recrystallization Inhibition of Ice-Binding Proteins and Bioinspired Synthetic Mimics In Extreme Environments Público Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/2v23vv457

Abstract

While water offers many unique and beneficial properties, the formation of ice can be detrimental for a variety of applications, such infrastructure, organ transplantation, aviation and energy surfaces, food preservation, space exploration. However, current ice prevention methodologies often rely on large quantities of potentially toxic small molecules (e.g., glycerol or dimethyl sulfoxide) to take advantage of the colligative effects they provide. Materials that prevent ice formation at lower concentrations through more robust mechanisms offer a revolutionary advancement for long-term economic and environmental effects on a wide variety of industries that must think about ice prevention.

Ice-binding proteins (IBPs) are a unique subset of natural proteins with the ability to prevent ice growth by increasing the energy for expansion of ice at the water-ice interface through the kelvin effect at nanomolar concentrations. However, proteins are expensive to produce and are known to restructure in non-physiological environments. Thus understanding the limits of IBP applicability as well as the crucial components for maximum efficacy are necessary to move towards synthetic replicates for large-scale applications. To mimic IBPs, it’s important to understand the functional and structural characteristics.

The main objectives of this research were (1) to use native IBPs as a biological template to create synthetic polymers that mimic ice prevention activities of IBPs, (2) to investigate homo-polypeptides of functional groups responsible for IBP ice prevention, and (3) to test a full synthetic replicate for performance and stability in non-physiological environments to act as an alternative to IBPs.

In the first study, 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.

In the second study, the ability of an ice-binding protein (IBP) from Marinomonas primoryensis (MpIBP) to influence ice crystal growth and structure in non-physiological 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. Results illustrate that MpIBP was stable between pH 6 – 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 mean largest grain size (MLGS) of ice crystals up to ~ 70% 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 non-physiological materials science and engineering applications.

In a third study, single amino acids and homo polypeptides based on IBP ice-binding residues were investigated for their ability to influence ice-crystal growth in phosphate buffered saline (PBS) using a modified splat assay to assess ice recrystallization inhibition (IRI), a measure of ice crystal growth inhibition. In PBS, the IRI activity of poly(threonine) at concentrations between 0.1-10 mg/ml reduced the mean largest grain size by ~75%. Based on its performance, threonine was chosen as a template to create a synthetic polymer mimic, poly(2-hydroxypropyl methacrylamide) (pHPMA). Poly(threonine) and pHPMA were investigated for their ability to influence ice-crystal growth in phosphate buffered saline (PBS), PBS solutions with alkaline pH (8-13), and 151.5 mM salt solutions with divalent cations relevant to infrastructure (CaCl2 and MgCl2). Overall, pHPMA reduced the mean largest grain size by ~60-75% at concentrations from 0.01-10 mg/ml, as well as inhibited ice growth by 80% at 10 mg/ml in pH 13 solution, demonstrating a viable option for mitigating ice growth in highly alkaline cementitious environments.

Considering that IBPs are an exemplary class of proteins with an ability to prevent ice formation in physiological environments, they offer a potentially disruptive model for creating bioinspired synthetic alternatives for ice growth mitigation in non-physiological environments for a variety of applications. It was shown that not only are IBPs prohibited by cost for application in non-native environments, but also by longevity. The crucial aspects of IBPs for their effect on ice growth inhibition i.e., ice-binding residues, structure-function relationship, were investigated, and it has been demonstrated that primary structure combined with ice-interactive residues is a crucial factor when considering design for ice recrystallization inhibitory materials. These results demonstrate that the ice-inhibition capabilities of future innovative bioinspired materials can be improved through incorporating amphipathic pendant groups, as demonstrated by the performance of poly(threonine) and pHPMA in non-physiological alkaline environments.

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