Protein/Ice Interaction: High-Resolution Synchrotron X-ray Diffraction Differentiates Pharmaceutical Proteins from Lysozyme

Bakul Bhatnagar, Boris Zakharov, Alexander Fisyuk, Xin Wen, Fawziya Karim, Kimberly Lee, Yurii Seryotkin, Mashikoane Mogodi, Andy Fitch, Elena Boldyreva, Anastasia Kostyuchenko, Evgenyi Shalaev

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

Protein/ice interactions are investigated by a novel method based on measuring the characteristic features of X-ray diffraction (XRD) patterns of hexagonal ice (Ih). Aqueous solutions of four proteins and other solutes are studied using high-resolution synchrotron XRD. Two pharmaceutical proteins, recombinant human albumin and monoclonal antibody (both at 100 mg/mL), have a pronounced effect on the properties of ice crystals, reducing the size of the Ih crystalline domains and increasing the microstrain. Lysozyme (100 mg/mL) and an antifreeze protein (1 mg/mL) have much weaker impact on Ih. Neither of the proteins studied exhibit preferred interactions with specific crystalline faces of Ih. It is proposed that the pharmaceutical proteins interact with ice crystals indirectly by accumulating in the quasi-liquid layer next to ice crystallization front, rather than directly, via a sorption on ice crystals. This is the first report, to the best of our knowledge, of major difference in the protein/ice interaction between non-antifreeze proteins. Another important finding is a detection of a second (minor) population of ice crystals, which is tentatively identified as a high-pressure form of ice, possibly IceIII or IceIX. This finding highlights a potential role of mechanical stresses in freeze-induced destabilization of proteins.

Original languageEnglish
Pages (from-to)5690-5699
Number of pages10
JournalJournal of Physical Chemistry B
Volume123
Issue number27
DOIs
Publication statusPublished - 17 Jun 2019

Keywords

  • ICE RECRYSTALLIZATION
  • ANTIFREEZE PROTEINS
  • SOLUTE CRYSTALLIZATION
  • STABILITY
  • WATER
  • ADSORPTION
  • KINETICS
  • BINDING
  • PHASE
  • LINE

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