Nanocrystallites Modulate Intermolecular Interactions in Cryoprotected Protein Solutions

Studying protein interactions at low temperatures hasimportantimplications for optimizing cryostorage processes of biological tissue,food, and protein-based drugs. One of the major issues is relatedto the formation of ice nanocrystals, which can occur even in thepresence of cryoprotectants and can lead to protein denaturation.The presence of ice nanocrystals in protein solutions poses severalchallenges since, contrary to microscopic ice crystals, they can bedifficult to resolve and can complicate the interpretation of experimentaldata. Here, using a combination of small- and wide-angle X-ray scattering(SAXS and WAXS), we investigate the structural evolution of concentratedlysozyme solutions in a cryoprotected glycerol-water mixturefrom room temperature (T = 300 K) down to cryogenictemperatures (T = 195 K). Upon cooling, we observea transition near the melting temperature of the solution (T & AP; 245 K), which manifests both in the temperaturedependence of the scattering intensity peak position reflecting protein-proteinlength scales (SAXS) and the interatomic distances within the solvent(WAXS). Upon thermal cycling, a hysteresis is observed in the scatteringintensity, which is attributed to the formation of nanocrystallitesin the order of 10 nm. The experimental data are well described bythe two-Yukawa model, which indicates temperature-dependent changesin the short-range attraction of the protein-protein interactionpotential. Our results demonstrate that the nanocrystal growth yieldseffectively stronger protein-protein attraction and influencesthe protein pair distribution function beyond the first coordinationshell.

Automated summary

Studying protein interactions at low temperatures is crucial for optimizing cryostorage processes. The formation of ice nanocrystals, even in the presence of cryoprotectants, can lead to protein denaturation. This presents challenges in resolving and interpreting experimental data.