Molecular nucleation mechanisms and control strategies for crystal polymorph selection

Area of Science:

• Biophysics
• Structural Biology
• Materials Science

Background:

• Condensed protein phases are linked to human diseases (e.g., Alzheimer's) but are also vital for structural biology and pharmaceuticals.
• Controlling protein polymorphs requires understanding nucleation pathways and selection mechanisms.
• Current understanding of macromolecular phase nucleation and its influence on polymorph selection is incomplete.

Purpose of the Study:

• To elucidate the molecular-level nucleation pathways of protein crystals.
• To understand the mechanisms governing polymorph selection during protein phase formation.
• To demonstrate control over protein polymorph formation for scientific and industrial applications.

Main Methods:

• Time-resolved cryo-transmission electron microscopy (cryo-TEM) was used to image crystal nucleation.
• The nucleation of glucose isomerase crystals was studied to identify pathways leading to crystalline and gelled states.
• Site-directed mutagenesis and gel seeding were employed to control polymorph formation.

Main Results:

• Polymorph selection is determined at the earliest stages of structure formation, based on specific building blocks for each space group.
• Nucleation occurs via oriented attachments between subcritical clusters with inherent crystallinity, not from a dense liquid precursor.
• Selective formation of desired polymorphs was achieved through targeted mutagenesis and gel seeding.

Conclusions:

• The study provides a molecular-level understanding of protein nucleation pathways and polymorph selection.
• Findings challenge existing models of protein nucleation, highlighting oriented attachment of crystalline clusters.
• The insights enable control over macromolecular phase transitions, advancing protein-based drug delivery and crystallography.