Freezing is a ubiquitous process in nature and pharmaceutical manufacturing processes. In the present study, several classes of proteins (isoelectric points, pIs 4.7 to 11.4) were investigated, including an insect antifreeze protein (AFP), model proteins (recombinant human albumin, lysozyme), and a pharmaceutical protein (monoclonal antibody). AFPs are found in many cold-adapted organisms and are well-known for their ability to inhibit ice growth through binding to specific ice crystal surfaces. AFPs from insects generally exhibit higher antifreeze activity than those from other organisms. However, there have been limited studies on insect AFP-ice interactions, which impedes our understanding of AFP-ice interaction mechanisms and limits their application. We present a new method for investigation of protein-ice interaction, which is based on measuring characteristic features of X-ray diffraction (XRD) patterns of hexagonal ice (Ih). Protein-ice interaction can be direct (sorption of protein on ice crystals), and / or indirect (partitioning of protein molecules into the quasi-liquid layer), thereby exhibiting different “signatures” in the Ih XRD patterns. The experiments were performed using high-resolution synchrotron X-ray diffraction (SXRD) at ID22 beamline at the ESRF, which is optimized to eliminate the instrumental broadening of diffraction lines and reduce the preferred orientation effects, thereby facilitating the quantitative analysis of the X-ray diffraction data. SXRD patterns of ice were obtained at 100 and 228 K in the absence and presence of the proteins. AFP inhibited growth of ice Ih crystals in directions corresponding to hkl 002, 110, 200, and 105, suggesting a direct AFP-ice interaction. Peak analyses also revealed that the AFP decreased ice crystal size and increased strain. Preliminary data analysis suggests a possible contribution of the pI of the protein on phase behavior of Ih. In this presentation, we present a new approach to investigate the protein-ice system, which could improve understanding of ice-protein interactions (AFPs, non-AFPs), and provide insights into degradation and stabilization mechanisms of frozen therapeutic proteins.

Probing protein-ice interactions using high resolution synchrotron X-ray diffraction

255th National Meeting (2018)

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