Nanocrystallites Modulate Intermolecular Interactions in Cryoprotected Protein Solutions

Area of Science:

• Biophysics
• Materials Science

Background:

• Low-temperature protein interactions are vital for cryopreservation of biologicals and drugs.
• Ice nanocrystal formation during cooling can denature proteins and complicate experimental analysis.

Purpose of the Study:

• To investigate the structural evolution of concentrated lysozyme solutions in a cryoprotected glycerol-water mixture from room temperature to cryogenic temperatures.
• To understand the impact of ice nanocrystal formation on protein-protein interactions and solution structure.

Main Methods:

• Utilized small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) to study lysozyme solutions.
• Cooled samples from 300 K to 195 K and performed thermal cycling.
• Analyzed scattering data using the two-Yukawa model.

Main Results:

• Observed a transition near the solution's melting temperature (≈245 K) affecting protein-protein interactions and solvent structure.
• Detected hysteresis in scattering intensity upon thermal cycling, attributed to 10 nm nanocrystallite formation.
• The two-Yukawa model fit indicated temperature-dependent changes in protein-protein attraction.

Conclusions:

• Nanocrystal growth enhances protein-protein attraction, influencing the protein pair distribution function.
• Understanding these low-temperature structural changes is key for improving cryopreservation strategies.
• SAXS and WAXS provide valuable insights into protein behavior under cryogenic conditions.